Effects on recovery during, acidosis in cardiac myocytes overexpressing CaMKII

Abstract

Recovery of intracellular Ca transients and fractional shortening during late phase acidosis are suggested to be associated with CaMKII-dependent processes of which phospholamban (PLB) phosphorylation may play an important role. To test whether increased expression levels of CaMKII may further enhance recovery, we investigated myocytes from CaMKII delta(C) transgenic (TG) mice (cytosolic localized CaMKII) having heart failure vs. wildtype littermates (WT). Furthermore, mouse and rabbit myocytes overexpressing CaMI:I delta(C) using adenovirus-mediated gene transfer (vs. LacZ control) were investigated. Fractional shortening (% vs. resting cell length, % RCL was assessed during control conditions (pH 7.4) and during acidosis (pH 6.5). Ca transients were measured using fluo-3 (Delta F/F-0, 10 mu M). In WT mouse myocytes, fractional shortening clearly recovered by 90% from 4.6 +/- 0.6 to 7.2 +/- 0.7% RCL during late acidosis. In parallel, Ca transients increased from 2.01 +/- 0. 11 to 2.33:L 0. 15 Delta F/F-0. When blocking CaMKII (KN-93, I mu M), recovery of Ca transients and shortening could be completely abolished, In contrast, in CaMKII delta(C) TG mouse myocytes shortening recovered only by 32% from 3.4 +/- 0.6 to 4.4 +/- 0.5% RCL (P< 0.05 vs. WT using ANOVA). In parallel, Ca transients increased only slightly from 1.75 +/- 0.15 to 1.84 +/- 0.13 Delta F/F-0 (P< 0.05 vs. WT using ANOVA). In accordance, SR Ca content (measured by caffeine contractures, 10 mM) in WT significantly increased during late acidosis but not in CaMKII delta(C) TG mice. In contrast, in mouse and rabbit myocytes overexpressing CaMKII delta(C) by means of adenovirus-mediated gene transfer, recovery of fractional shortening and Ca transients was not impaired during late acidosis but even slightly improved vs. LacZ control (P<0.05 vs. CaMKII delta(C) using ANOVA for mouse and rabbit myocytes). This was associated with significantly increased SR Ca content during late acidosis in CaMKII delta(C) as compared to LacZ. CaMKII-dependent PLB Thr- 17 phosphorylation, contributing to increased SR Ca uptake, was significantly increased in CaMKII delta(C) transfected rabbit myocytes vs. LacZ in the light of unchanged SR Ca ATPase and PLB protein expression. CaMKII inhibition completely prevented recovery of all parameters in both CaMKII delta(C) and LacZ. In summary and in contrast to our initial hypothesis, we showed for the first time that TG CaMKII delta(C) overexpression (i.e., chronic overexpression) in mice with heart failure clearly resulted in impaired recovery associated with impaired SR Ca loading during late acidosis vs. WT. This may be due to decreased SR Ca ATPase and PLB expression as reported previously. In contrast, adenovirus-mediated gene transfer of CaMKII delta(C) in mouse and rabbit myocytes (i.e., acute overexpression) did not result in impaired but even slightly improved recovery associated with increased SR Ca load during late acidosis as compared to LacZ. This most likely was due to higher PLB Thr- 17 phosphorylation in CaMKII delta(C) myocytes. In conclusion, possible beneficial effects by therapeutical CaMKII delta(C) stimulation on the ability to recover from acidosis may be challenged by altered expression levels of its target proteins and should be carefully considered. (c) 2007 Elsevier Inc. All rights reserved.