CTR; (b) Immunoprecipitation experiments and densitometric analysis (on the right) indicating higher acetylation level of SERCA2 after SAHA treatment (= 5). to ameliorate SERCA2 efficiency. and = 7). MannCWhitney 0.005 vs. CTR; (b) Immunoprecipitation experiments and densitometric analysis (on the CC-671 right) indicating higher acetylation level of SERCA2 after SAHA treatment (= 5). MannCWhitney 0.005 vs. CTR; (c) Western blot analysis showing the expression of phosphorylated phospholamban (Phospho-PLB, Ser16) compared to total phospholamban (PLB) in adult rat CMs after SAHA treatment (= 7). CC-671 Co-immunoprecipitation experiments revealed that SAHA increased SERCA2 acetylation level (Physique 1b and Physique S1), without inducing significant changes in SERCA2 protein expression (Physique 1a). The ratio of phosphorylated phospholamban (PLB)/total phospholamban (Physique 1c) remained unchanged. To investigate whether increased acetylation could also affect SERCA2 functional properties, ATPase activity was measured on microsomes [19] isolated from both CTR and CTR+SAHA CMs. HDAC inhibition resulted in an increase of ATPase activity when microsomes were exposed to 10 M calcium concentration (corresponding to pCa5) [20,21] (Physique 2a). Open in a separate window Physique 2 Effect of SAHA treatment on SERCA2 ATPase activity evaluated in cardiomyocytes isolated from adult CC-671 rat hearts and HL-1 cells. (a) ATPase activity assay performed on microsomes isolated from adult rat CMs either untreated or treated with SAHA at pCa5. Experiments were performed on 3 impartial CM sets Mouse monoclonal to CER1 per group and repeated twice. Unpaired Students 0.005 vs. CTR; (b) ATPase activity assay performed on microsomes isolated from HL-1 cells at different pCa. Each point represents the mean SEM of at least 4 impartial experiments; Two-way ANOVA followed by Sidaks multiple comparison: * 0.05 vs. SAHA pCa6; # 0.05 vs. SAHA pCa5. All data are presented as mean SEM. In order to confirm our result on SERCA2 functional properties, we decided to perform an additional set of experiments measuring ATPase activity on microsomes isolated from HL-1 cells, derived from the AT-1 mouse atrial cardiomyocyte tumor lineage. These cells partially maintain an adult cardiac phenotype and are able to contract [22]. The calcium-dependence of ATPase activity on HL-1 cells, either untreated (CTR) or treated for 90 min with 2.5 M SAHA, was analyzed at different calcium concentration (from pCa8 to pCa5). ATPase activity of microsomes extracted from HL-1 cells was increased after SAHA treatment in comparison with CTR and the difference reached statistical significance when microsomes were exposed to 1 and 10 M calcium concentration (corresponding to pCa6 and pCa5, respectively; Physique 2b). 2.2. Effect of SAHA Treatment on Calcium Transients and Cell Mechanics in CMs Isolated from Adult Rat Hearts We then investigated whether SAHA treatment affected CM functional parameters that directly depend on SERCA2 activity, namely calcium transients and CC-671 cell contractility. The amplitude and the time to peak (TTP) of the calcium transient were comparable in CTR and CTR+SAHA groups, while the rate of cytosolic calcium clearing was significantly higher in SAHA-treated cardiomyocytes (Physique 3a,c). Specifically, SAHA induced a 21% decrease in the time constant tau, as well as a significant reduction in the time to 10%, 50% and 90% of fluorescence signal decay (BL10, BL50, BL90; Physique 3c). Consistent with this obtaining, SAHA also affected CM mechanics during the re-lengthening phase, as documented by the significant increase in the maximal rate of re-lengthening (+dl/dtmax, approximately 16%) associated with a decrease in the time to 10%, 50% and 90% of re-lengthening (Physique 3b,d). Conversely, the average diastolic sarcomere length, the fraction of shortening and the maximal rate of shortening were comparable.