Contractile arrest increases sarcoplasmic reticulum calcium uptake and SERCA2 gene expression in cultured neonatal rat heart cells.

Abstract

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 (Cai) transients were measured by use of indo-1 during (1) spontaneous twitches, (2) contractures induced by rapid application of caffeine (CafC, with and without Nao), 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 t1/2 for [Ca]i decline when both the SR Ca uptake and Na-CaX were prevented was the same for control and VA cells (approximately 20 seconds), indicating unaltered slow Ca transport systems. Similarly, there was no significant difference in the t1/2 of CafC when Na-CaX was the main mechanism responsible for [Ca]i decline (t1/2 approximately 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 t1/2, 0.84 +/- 0.05 seconds; VA t1/2, 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).(ABSTRACT TRUNCATED AT 250 WORDS)