Exogenous Ca2+-ATPase isoform effects on Ca2+ transients of embryonic chicken and neonatal rat cardiac myocytes.

Sarco-endoplasmic reticulum Ca2+-ATPase from fast skeletal (SERCA1) or cardiac muscle (SERCA2a) was expressed in embryonic chicken and neonatal rat cardiac myocytes by adenovirus vectors, with c-myc tags on both constructs to compare expression and distinguish exogenous from endogenous SERCA2a in myocytes. Expression of the two isoforms was similar (approximately 3-fold higher than endogenous SERCA). However, SERCA1 activity was 2-fold greater than SERCA2a activity, due to intrinsic differences in turnover rates. Activation of both exogenous SERCA isoforms by Ca2+ was displaced to slightly lower [Ca2+], suggesting that the overexpressed isoforms were independent of phospholamban. In fact, phospholamban and calsequestrin expression were unchanged. Decay time constants of cytosolic Ca2+ transients from cells overexpressing SERCA1 were reduced by 30–40 % and half-widths by 10–15 % compared to controls. SERCA2a overexpression produced much less acceleration of transients in chick than in rat, and less acceleration than SERCA1 overexpression in either species. There was no significant change in resting [Ca2+], peak amplitudes, or in the amount of Ca2+ releasable by caffeine from overexpression of either SERCA isoform. However, the amplitudes of the transients increased with SERCA1 overexpression when pacing frequency limited refilling of the sarcoplasmic reticulum. It is concluded that total SERCA transport velocity has a primary effect on the decay phase of transients. Transport velocity is affected by SERCA isoform turnover rate, temperature, and/or SERCA copy number. Active transport of cytosolic calcium into intracellular stores by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) is important in regulating calcium signalling. In skeletal and cardiac muscle, SERCA is especially critical, as transport of Ca2+ into the sarcoplasmic reticulum (SR) is required to terminate transient rises in cytosolic Ca2+ that couple membrane excitation to contractile activation, as well as to replenish emptied Ca2+ stores (Carsten, 1964; Fanburg et al. 1964; Inesi et al. 1964). Consistent with this role, specific inhibition of SERCA by thapsigargin drastically reduces cytosolic Ca2+ transients, tension development and kinetics of relaxation in cardiac myocytes, in the absence of changes in action potential (Kirby et al. 1992). Furthermore, mice heterozygous for a null mutation in the cardiac isoform of SERCA have impaired cardiac function (Periasamy et al. 1999). Relevant to these findings is the observation that SERCA mRNA and protein levels decrease in end-stage heart failure (Gwathmey et al. 1987; Mercadier et al. 1990; Takahashi et al. 1992; Hasenfuss et al. 1994), and the suggestion that defective expression of the ATPase may play a pathogenic role (Morgan et al. 1990; Hasenfuss et al. 1994, 1997; Schmidt et al. 1998). To date, there are three known SERCA genes with several splice variants. SERCA1 is the endogenous isoform of fast-twitch skeletal muscle, while SERCA2a is found in slow-twitch skeletal and cardiac muscle (Brandl et al. 1986; Kaprielian & Fambrough, 1987; Zarain-Herzberg et al. 1990). SERCA3 is mainly expressed in epithelial and endothelial cells (Anger et al. 1994), and platelets (Bokkala et al. 1995). Expression of exogenous SERCA1 or SERCA2a genes in cardiac myocytes has been recently achieved by transfecting cultured cells (Inesi et al. 1997, 1998; Hajjar et al. 1997; Giordano et al. 1997; Sumbilla et al. 1999), or by developing transgenic mice (He et al. 1997; Loukianov et al. 1998; Baker et al. 1998). Although the amounts of exogenous gene expression varied among studies, it was reported that increases in SERCA levels produced accelerated decay phases in cytosolic calcium transients. Presently, however, it is not clear whether expression of exogenous SERCA in cardiac myocytes affects cytosolic Ca2+ transients by increasing the SR calcium load or by accelerating the velocity of the transients by SR ATPase activity. It is also uncertain whether the Ca2+ transients may be affected in a different way by expression of exogenous SERCA1 or SERCA2a, since the two isoforms were shown to have different Ca2+ transport turnover rates (Sumbilla et al. 1999). We have now carried out a systematic study of SERCA isoforms expressed in cultured embryonic chicken and neonatal rat cardiac myocytes. Viral vectors enabled us to target 100 % of cultured cells with controlled, reproducible SERCA expression 3- to 4-fold greater than controls, and higher than previously obtained by other laboratories (1·2-fold, He et al. 1997; 2-fold, Hajjar et al. 1997; 2·5-fold, Loukianov et al. 1998; 1·5-fold, Baker et al. 1998). We studied Ca2+ transients using the fluorescent indicators fluo-4 and fura-2, under conditions of single or repetitive stimulation. We also measured total Ca2+ released by caffeine from intracellular compartments, and determined the time course of store refilling during restitution protocols. Finally, we discuss variables which might account for differences in the effects of SERCA overexpression observed by ourselves and others.