Developmental changes of calcium transients and contractility during the cultivation of rat neonatal cardiomyocytes

Neonatal cardiomyocytes of the rat were investigated (a) by Confocal Laser Scanning Microscopy (CLSM) using the Ca(2+)-sensitive dye fluo-3/AM to measure calcium transients, and (b) by a Laser Doppler Microscope (LSC-1) to obtain data of the cell culture's contractility. Our experiments resulted in: (1) About 20% of the freshly prepared cardiomyocytes exhibited spontaneous but not rhythmically appearing calcium transients. None of these cells was found to be active mechanically. The remainder of 80% showed neither calcium transients nor cell movements. (2) At the latest after four days of cultivation, the cells showed spontaneous calcium transients of constant frequency and concomitant contractions. (3) During the cultivation, spontaneous Ca2+ transients became steeper and shorter. The time course of the calcium transient is abbreviated by a factor of at least two in cells after four days when compared with cardiac cells after one day of cultivation. (4) Addition of 100 nM ryanodine caused an increase of the cytosolic calcium concentration and a decrease of the amplitude of the Ca2+ transients. This effect became more significant with increasing time of cultivation and ran parallel to a decrease of the cell's contractility. (5) Addition of 1 microM thapsigargin yielded a similar increase of the cytosolic calcium concentration and a decrease of the Ca2+ peak accompanied by a smaller lowering of the contractility (in comparison with the mentioned influence of ryanodine). The effects of thapsigargin were practically independent of the time of cultivation.