Comparison of SERCA1 and SERCA2a expressed in COS-1 cells and cardiac myocytes

Abstract

Cultured COS-1 cells, as well as chicken embryonic and neonatal rat cardiac myocytes, were infected with recombinant adenovirus vectors to define limiting factors in the expression and Ca2+ transport function of recombinant sarcoplasmic-endoplasmic reticulum Ca(2+) (SERCA) isoforms. Titration experiments showed that all COS-1 cells and myocytes in culture could be infected by an adenovirus titer of 10 plaque-forming units (pfu) per seeded cell. Raising the adenovirus titer further yielded higher protein expression up to an asymptotic limit for functional, membrane-bound SERCA protein. The asymptotic behavior of SERCA expression was not transcription related but was due to posttranscriptional events. The minimal (-268) cardiac troponin T (cTnT) promoter was a convenient size for adenovirus vector construction and manifested tight muscle specificity. However, its efficiency was lower than that of the nonspecific cytomegalovirus (CMV) promoter. At any rate, identical maximal levels of SERCA expression were obtained with the CMV and the cTnT promoter, as long as the viral titer was adjusted to compensate for transcription efficiency. A maximal threefold increase of total SERCA protein expression over the level of the endogenous SERCA of control myocytes was reached (a sevenfold increase compared with the endogenous SERCA of the same infected myocytes due to reduction of endogenous SERCA after infection). In contrast with previous reports [Ji et al. Am. J. Physiol. 276 (Heart Circ. Physiol. 45): H89-H97, 1999], a higher kinetic turnover was demonstrated for the SERCA1 compared with the SERCA2a isoform as shown by a 5.0- versus 2.6-fold increase in calcium uptake rate accompanying maximal expression of recombinant SERCA1 or SERCA2a, respectively. This information is deemed necessary for studies attempting to modify myocardial cell function by manipulation of SERCA expression.

Variation in expression of calcium-handling proteins is associated with inter-individual differences in mechanical performance of rat (Rattus norvegicus) skeletal muscle

“…High ryanodine receptor activity would require increased rates of Ca 2+ resequestration into the sarcoplasmic reticulum to decrease relaxation times and thereby maintain muscle performance over time. High ryanodine receptor activity may therefore be incompatible with high SERCA2 concentrations, because our results and those of others (Sumbilla et al, 1999) indicate that elevated SERCA2 concentrations slow muscle relaxation. Hence, there is a functional trade-off between ryanodine receptor activity and both SERCA 1 and 2 isoforms.…”

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.…”

“…We used adenovirus-mediated gene transfer of SERCA1a into isolated rabbit ventricular cardiomyocytes. SERCA1a is a splice transcript of the SERCA1 gene (expressed in adult fast-twitch skeletal muscle) that has faster Ca 2ϩ transport kinetics 11 and might achieve higher levels of SERCA activity than equivalent upregulation of SERCA2a. 12 Rabbit ventricular myocytes were used because excitationcontraction coupling in rabbit myocardium is similar to that in humans.…”

SERCA overexpression reduces hydroxyl radical injury in murine myocardium

“…SERCA-overexpressed mice (transgenic, TG) are mice expressing the (skeletal) SERCA1a isoform (2.5-fold increase in the total amount of SERCA and an approximately twofold increase in SR Ca 2ϩ uptake function) (18). We chose the SERCA1a-overexpressed mice for this proof-of-principle study; SERCA1a possesses faster Ca 2ϩ transport kinetics, but unaltered Ca 2ϩ affinity, pH response, and PLB affinity, and therefore possible positive effects of enhanced SERCA activity (26) would be most pronounced. SERCA2a heterozygous knock-out mice (HET) have a ϳ40% reduction in expression of SERCA2a, resulting in a decreased SR calcium reuptake (10).…”