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 heating 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 (≃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 )≃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 slow Ca transport systems.