CONTRACTILE ARREST INCREASES SARCOPLASMIC-RETICULUM CALCIUM-UPTAKE AND SERCA2 GENE-EXPRESSION IN CULTURED NEONATAL RAT-HEART CELLS

We developed protocols with intact cultured neonatal rat myocytes to directly evaluate the function of the sarcoplasmic reticulum (SR) Ca-ATPase (or SERCA2), Na-Ca exchange (Na-CaX), and slow Ca transport systems (mitochondria and sarcolemmal Ca-ATPase). Spontaneously beating control cells were compared with cells cultured for 2 days in the presence of verapamil (verapamil-arrested cells, VA). Intracellular calcium (Ca-i) transients were measured by use of indo-1 during (1) spontaneous twitches, (2) contractures induced by rapid application of caffeine (CafC, with and without Na-o), and (3) twitches induced by brief depolarizations with high [K](o) solution (K-twitches). We also measured mRNA levels for the SR Ca-ATPase and Na-CaX in the same experimental preparations. The t(1/2) for [Ca](i) decline when both the SR Ca uptake and Na-CaX were prevented was the same for control and VA cells (approximate to 20 seconds), indicating unaltered slow Ca transport systems. Similarly, there was no significant difference in the t(1/2) of CafC when Na-CaX was the main mechanism responsible for [Ca](i) decline (t(1/2)approximate to 1.5 seconds), indicating unaltered Na-CaX. Conversely, we found nearly a twofold increase in the rate of [Ca](i) decline during K-twitches (control t(1/2), 0.84+/-0.05 seconds; VA t(1/2), 0.48+/-0.06 second; P<.001), indicating an increase in SR Ca-pumping activity in VA cells. This was also reflected by a 56% increase in the peak [Ca](i) reached during CafC used to assess maximal SR Ca content (427+/-49 nmol/L in control versus 665+/-75 nmol/L in VA cells). In agreement with these functional effects, we found no change in Na-CaX mRNA levels but a marked upregulation of both the SERCA2 mRNA and protein levels in VA cells (to 166+/-10% and 164+/-20%, respectively). Thus, verapamil arrest induced an increase in SR Ca uptake (and SERCA2 expression) without affecting the Na-CaX activity (or expression) or the dow Ca transport systems.