EFFECTS OF THAPSIGARGIN AND CYCLOPIAZONIC ACID ON INTRACELLULAR CALCIUM ACTIVITY IN NEWBORN RAT CARDIOMYOCYTES DURING THEIR DEVELOPMENT IN PRIMARY CULTURE

The effects of specific inhibitors of sarcoplasmic reticulum (SR) calc ium ATPase, thapsigargin (TG), and cyclopiazonic acid (CPA) were inves tigated on the resting and transient levels of intracellular free calc ium concentrations recorded in Indo-1-loaded ventricular myocytes of n ewborn rat heart in primary culture. The calcium transients were induc ed by caffeine (10 mM) or high potassium (100 mM) solutions. In 2 day- as in 7-day-old cultured cells, the calcium transients induced by 10 mM caffeine were blocked dose dependently by TG and CPA. The dose-resp onse curves suggest that TG was more efficient than CPA and that both drugs were more efficient in 7-day- than in 2-day-old cells. The calci um transients induced by 100 mM K+ were also strongly inhibited by the se agents. The lack of effect on sarcolemmal calcium currents, as show n by whole-cell patch-clamp experiments, suggests that these drugs aff ect only SR function. In cells exhibiting spontaneous activity, the as sociated calcium transients were not affected by TG or CPA at the begi nning of the culture (2-day-old cells) but were fully blocked at the e nd (7-day-old cells). These results confirm that TG and CPA specifical ly inhibit the cardiac SR Ca2+ pump without affecting the sarcolemmal calcium current. Their blocking effect of the calcium transients as a function of the developmental stage of neonatal cardiac cells in cultu re suggests an increasing role of the SR in the regulation of intracel lular calcium. This argues for developmental changes of the SR through the differentiation and maturation of newborn cardiomyocytes at the e arly stage of the postnatal life, leading to a predominant role of the SR in excitation-contraction coupling mechanisms in adult cells.