4-hydroxytamoxifen does not deteriorate cardiac function in cardiomyocyte-specific MerCreMer transgenic mice

Conditional, cell-type-specific transgenic mouse lines are of high value in cardiovascular research. A standard tool for cardiomyocyte-restricted DNA editing is the αMHC-MerCreMer/loxP system. However, there is an ongoing debate on the occurrence of cardiac side effects caused by unspecific Cre activity or related to tamoxifen/oil overload. Here, we investigated potential adverse effects of DNA editing by the αMHC-MerCreMer/loxP system in combination with a low-dose treatment protocol with the tamoxifen metabolite 4-hydroxytamoxifen (OH-Txf). αMHC-MerCreMer mice received intraperitoneally OH-Txf (20 mg/kg) for 5 or 10 days. These treatment protocols were highly efficient to induce DNA editing in adult mouse hearts. Multi-parametric magnetic resonance imaging revealed neither transient nor permanent effects on cardiac function during or up to 19 days after 5 day OH-Txf treatment. Furthermore, OH-Txf did not affect cardiac phosphocreatine/ATP ratios assessed by in viv

Systematic Analysis Reveals Elongation Factor 2 and α-Enolase as Novel Interaction Partners of AKT2

<div><p>AKT2 is one of the three isoforms of the protein kinase AKT being involved in the modulation of cellular metabolism. Since protein-protein interactions are one possibility to convey specificity in signal transduction, we performed AKT2-protein interaction analysis to elucidate their relevance for AKT2-dependent cellular functions. We identified heat shock protein 90 kDa (HSP90), Cdc37, heat shock protein 70 kDa (HSP70), 78 kDa glucose regulated protein (GRP78), tubulin, GAPDH, α-enolase and elongation factor 2 (EF2) as AKT2-interacting proteins by a combination of tandem affinity purification and mass spectrometry in HEK293T cells. Quantitative MS-analysis using stable isotope labeling by amino acids in cell culture (SILAC) revealed that only HSP90 and Cdc37 interact stably with AKT2, whereas the other proteins interact with low affinity with AKT2. The interactions of AKT2 with α-enolase and EF2 were further analyzed in order to uncover the functional relevance of these newly discovered binding partners. Despite the interaction of AKT2 and α-enolase, which was additionally validated by proximity ligation assay (PLA), no significant impact of AKT on α-enolase activity was detected in activity measurements. AKT stimulation via insulin and/or inhibition with the ATP-competitive inhibitor CCT128930 did not alter enzymatic activity of α-enolase. Interestingly, the direct interaction of AKT2 and EF2 was found to be dynamically regulated in embryonic rat cardiomyocytes. Treatment with the PI3-kinase inhibitor LY294002 before stimulation with several hormones stabilized the complex, whereas stimulation alone led to complex dissociation which was analyzed <i>in situ</i> with PLA. Taken together, these findings point to new aspects of AKT2-mediated signal transduction in protein synthesis and glucose metabolism.</p></div

Dynamic regulation of the AKT2/EF2-interaction in embryonic rat cardiomyocytes.

<p>A/D/G: PLA after inhibition of PI3-Kinase with LY294002 (1 h, 50 µM) and subsequent angiotensin II (A), IGF-1(D) and insulin (G) stimulation B/E/H: PLA after stimulation of the cells with angiotensin II (30 minutes, 100 nM) (B), IGF-1(10 minutes, 1 µg/ml) (E) and insulin (10 minutes, 1,75 µg/ml) (H). C/F/I: Quantitative data of two independent PLA-experiments for each stimulant. The number of signals/nuclei for the LY294002+ stimulant sample was set as 100%.</p

Results of PLA of AKT and EF2.

<p>A–D: PLA on HEK293T AKT2-NTag cells A/B: Control experiments with one primary antibody. C: Complete PLA. D: Quantitative analysis of two independent experiments. E–L: Results of PLA with AKT2 and AKT1 with EF2 in embryonic rat cardiomyocytes. E–H: PLA AKT2/EF2 E/F: Control experiments with one primary antibody. G: Complete PLA. H: Quantitative analysis. I–L: PLA AKT1/EF2 I/J: Control experiments with one primary antibody. K: Complete PLA. L: Quantitative analysis. The number of signals/nuclei in the PLA-samples was set as 100%. Controls are shown as percentage of corresponding full PLA.</p

Detail of representative MS-spectra for peptide ratios of a stably and a transiently bound protein after MBP and MAP.

<p>A: Light and heavy form of the peptide DAGTIAGLNVMR of HSP90α with a shifted ratio for both approaches. B: Light and heavy form of the peptide SDIDEIVLVGGSTR of GRP78 with a ∼1∶1 ratio after MBP and a shifted ratio after MAP.</p

Overview of proteins most frequently detected in six (I-VI) TAPs with AKT2-NTag.

<p>Listed are those proteins, which were identified in three to six experiments.</p><p>x: Protein was not detected.</p><p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066045#pone.0066045.s005" target="_blank">Table S1</a> shows a detailed table with all identified proteins of these six TAPs.</p

α-enolase activity assay.

<p>A: Activity of immuno-captured α-enolase (ENO1) from HEK293T cells after stimulation with insulin and AKT-inhibition with CCT128930. Data represent means ± SD of n = 7 experiments. B: Detection of immuno-captured α-enolase after α-enolase activity assay. After detection of α-enolase blots were incubated with anti-pan AKT antibody. Note that AKT was also detected after isolation of α-enolase by the α-enolase antibody.</p

The AKT/EF2 connection.

<p>AKT is indirectly involved in EF2-activation via mTOR, AMPK and EF2K. Our data reveal a direct interaction of AKT2 and EF2, which is dynamically regulated upon angiotensin II, IGF-1 and insulin stimulation.</p