5 that SR Ca2+ articles was unchanged by exogenous SERCA expression. 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 Mouse monoclonal to Caveolin 1 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 Trimetrexate terminate transient Trimetrexate rises in cytosolic Ca2+ that couple membrane excitation to contractile activation, as well as to replenish emptied Ca2+ stores (Carsten, 1964; Fanburg 1964; Inesi 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 1992). Furthermore, mice heterozygous for a null mutation in the cardiac isoform of SERCA have impaired cardiac function (Periasamy 1999). Relevant to these findings is the observation that SERCA mRNA and protein levels decrease in end-stage heart failure (Gwathmey 1987; Mercadier 1990; Takahashi 1992; Hasenfuss 1994), and the suggestion that defective expression of the ATPase may play a pathogenic role (Morgan 1990; Hasenfuss 1994, 1997; Schmidt 1998). To date, there are three known genes with several splice variants. is the endogenous isoform of fast-twitch skeletal muscle, while is found in slow-twitch skeletal and cardiac muscle (Brandl 1986; Kaprielian & Fambrough, 1987; Zarain-Herzberg 1990). is mainly expressed in epithelial and endothelial cells (Anger 1994), and platelets (Bokkala 1995). Expression of exogenous or genes in cardiac myocytes has been recently achieved by transfecting cultured cells (Inesi 1997, 1998; Hajjar 1997; Giordano 1997; Sumbilla 1999), or by developing transgenic mice (He 1997; Loukianov 1998; Baker 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 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 (12-fold, He 1997; 2-fold, Hajjar 1997; 25-fold, Loukianov 1998; 15-fold, Baker 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 Trimetrexate SERCA overexpression observed by ourselves and others. METHODS Adenovirus vectors Adenovirus vectors were constructed by pJM17 (Graham & Prevec, 1992) or Cre-lox recombination (Hardy 1997). Viruses Ad.Sr1 and Ad.Sr2a expressed chicken (Karin 1989) or (Campbell 1991), driven by a cytomegalovirus promoter and containing a c-tag (Evan 1985). Plaque purification and amplification were as described by Strock (1998) and Sumbilla (1999). Primary cell culture and infection Chicken embryonic cardiac myocytes were prepared from 8-day embryos and cultured in medium 199 with 5 % fetal calf serum, at 37C and 5 % CO2 (Inesi 1998). For fluorescence studies, 650 cells mm?2 were plated on collagen-coated coverslips. To obtain rat myocytes, 1-day-old rats were decapitated and the hearts were excised. Harvesting of cardiac tissue was carried out using protocols approved by the University of Maryland Institutional Animal Care and Use Committee. Myocytes were dissociated as described by Simpson (1989) and 250 cells mm?2 were plated on collagen-coated dishes or coverslips..