Abstract
A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase isoform 2 (SERCA2) gene, in which SERCA2 protein levels are reduced by approximately 35%, was used to investigate the effects of decreased SERCA2 level on intracellular Ca(2+) homeostasis and contractile properties in isolated cardiomyocytes. When compared with wild-type controls, SR Ca(2+) stores and Ca(2+) release in myocytes of SERCA2 heterozygous mice were decreased by approximately 40-60% and approximately 30-40%, respectively, and the rate of myocyte shortening and relengthening were each decreased by approximately 40%. However, the rate of Ca(2+) transient decline (tau) was not altered significantly, suggesting that compensation was occurring in the removal of Ca(2+) from the cytosol. Phospholamban, which inhibits SERCA2, was decreased by approximately 40% in heterozygous hearts, and basal phosphorylation of Ser-16 and Thr-17, which relieves the inhibition, was increased approximately 2- and 2.1-fold. These results indicate that reduced expression and increased phosphorylation of phospholamban provides compensation for decreased SERCA2 protein levels in heterozygous heart. Furthermore, both expression and current density of the sarcolemmal Na(+)-Ca(2+) exchanger were up-regulated. These results demonstrate that a decrease in SERCA2 levels can directly modify intracellular Ca(2+) homeostasis and myocyte contractility. However, the resulting deficit is partially compensated by alterations in phospholamban/SERCA2 interactions and by up-regulation of the Na(+)-Ca(2+) exchanger.
Highlights
In heart, muscle relaxation is largely dependent on the action of the sarcoplasmic reticulum (SR)1 Ca2ϩ ATPase (SERCA)
Cardiomyocytes from SERCA2 Heterozygous Hearts Have Lower Cytosolic Peak Ca2ϩ Transients—We have recently shown that disruption of one copy of the SERCA2 gene results in decreased SERCA2 mRNA (ϳ45%), protein (ϳ35%), and SR Ca2ϩ uptake (ϳ35%) and that these changes are associated in vivo with impaired cardiac performance [5]
Left ventricular myocytes isolated from wild-type and heterozygous hearts were loaded with Fura-2 AM, and the Ca2ϩ signals during electrical pacing at 0.25 Hz were measured
Summary
Gene targeting was used to delete part of the promoter, the transcript initiation site, and the first two coding exons of the SERCA2 gene [5]. Cardiac homogenates in assay buffer (100 g of total protein) with various concentrations (0.1–30 nM) of [3H]ryanodine (56.9 Ci/mmol, PerkinElmer Life Sciences) were incubated at 37 °C for 90 min in the presence and absence of 17 M cold ryanodine, and reactions were terminated by filtration. Measurement of [Ca2ϩ]i Transients and Contractile Parameters in Isolated Ventricular Myocytes—Adult left ventricular myocytes were isolated and resuspended in a physiological buffer at 37 °C for 15–20 min [2, 19, 20]. Half of the cells from each heart were used for mechanical studies, and the other half were used for measurements of intracellular free Ca2ϩ transient. Measurement of Naϩ-Ca2ϩ Exchanger Current Density—Naϩ-Ca2ϩ exchanger currents were recorded using whole-cell patch-clamp techniques as described previously [25,26,27]. Statistical Analyses—Results are expressed as mean Ϯ S.E. and statistically evaluated by the ANOVA test followed by the Student t test. p Ͻ 0.05 was considered to be the threshold for statistical significant
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