Isolation of cardiomyocytes from the adult mouse heart / 生理学报

ABSTRACT

In order to culture cardiomyocytes or to observe the contractile function of adult mouse cardiomyocytes, it is necessary to isolate high-yield and high-quality cardiomyocytes at first. The mouse was injected with heparin (5,000 IU/kg, i.p.) 20 min prior to the experimental protocol, then was sacrificed by cervical dislocation. The heart was excised and the aorta was cannulated rapidly. The cannulated heart was mounted on a Langendorff perfusion apparatus with constant flow and perfusion pressure was monitored. The initial perfusion pressure was maintained at 40 mmHg by regulating the flow rate. The heart was digested by 0.05 % crude collagenase I at 37 degrees C and the enzymatic digestion was terminated immediately when the perfusion pressure was lowered to 28 mmHg. The heart was then cut off the cannula and the atria and aorta dissected away. The ventricular tissue was chopped and the single myocyte was dispersed gently by a wide tipped pipette. The viability of freshly isolated cardiomyocytes was more than 70 %. The cardiomyocytes were kept in Joklik's minimum essential medium containing 1 % BSA and 10 mmol/L BDM, then extracellular calcium was restored step-wise to a final concentration of 1.25 mmol/L. The viability of cardiomyocytes reduced to (40-50) % after 4 h standing. More than 90 % of rod-shaped cardiomyocytes were quiescent and had visible cross striations and sharp edges. The amplitude of unloaded shortening in cardiomyocytes was (9.72+/-0.43) % during 1.0 Hz stimulation, (11.28+/-0.43) % at 2.0 Hz and (11.40+/-0.45) % at 5.0 Hz. These results indicate that high yield and high quality cardiomyocytes can be obtained. In addition, the standards of identifying cardiomyocyte quality are concise and are suitable to culture the cardiomyocytes or to study the physiological function of cardiomyocytes.