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

Abstract

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.

Limited functional and metabolic improvements in hypertrophic and healthy rat heart overexpressing the skeletal muscle isoform of SERCA1 by adenoviral gene transfer in vivo

“…In addition, densitometry analysis of Coomassie blue staining indicated that the total SERCA content increased 34 Ϯ 15%, and the overexpression of SERCA1 in whole hearts did not coincide with a measurable drop in endogenous SERCA2a levels. This contrasts with the earlier transgenic mouse model and our own cardiomyocyte data (5,30). In transgenic mice expressing SERCA1, endogenous SERCA2a expression was reduced by 50% and total SERCA levels increased by 2.5-fold (17,25,30).…”

“…Compared with the cardiac isoform (SERCA2a), SERCA1 is not regulated by phospholamban (44); it has a higher activity and a twofold greater calcium uptake relative to SERCA2a (5,19), and it is also more resistant to oxidative stress (61) and acidosis (63). Overexpressing this isoform in isolated rat cardiomyocytes and transgenic mice resulted in a two-to fourfold increase in the rates of Ca 2ϩ transport activity and increased tension development (5,19,25). In addition, isolated human cardiomyocytes from end-stage failing hearts overexpressing the SERCA1 isoform showed markedly improved contractile function (62).…”

Excessive Sarcoplasmic/Endoplasmic Reticulum Ca 2+ -ATPase Expression Causes Increased Sarcoplasmic Reticulum Ca 2+ Uptake but Decreases Myocyte Shortening

“…Here, the fast-twitch skeletal muscle SERCA1a was used because it has faster Ca 2ϩ transport kinetics versus SERCA2a. 11,16 Furthermore, SERCA1a transfection in cultured neonatal cardiomyocytes generated higher SERCA protein levels than parallel SERCA2a studies. 12 SERCA1a targets to intracellular membranes after adenovirus-mediated gene transfer into embryonic cardiac myocytes in a pattern identical to that of SERCA2a.…”

“…16 Briefly, cDNA of the chicken SERCA1a gene was expressed as a fusion protein with a c-terminal c-myc tag under control of the constitutively active cytomegalovirus promoter.…”

Advantages of Catheter-Based Adenoviral Delivery of Genes to the Heart for Studies of Cardiac Disease

“…Densitometry analysis of Coomassie blue stained gels indicated that the total SERCA content increased by 34 ± 15% in both banded and sham hearts, and endogenous SERCA2a expression levels were unaffected. Indeed, this level of expression is lower than the level reported for the SERCA1 transgenic mouse model and our own earlier cardiomyocyte data (Cavagna et al, 2000;Loukianov et al, 1998). In transgenic mice expressing SERCA1, total SERCA levels increased by 2.5-fold and endogenous SERCA2a expression were reduced by 50% (Huke et al, 2002;Lalli et al, 2001;Loukianov et al, 1998).…”