Loss of GRK2 in Cardiomyocytes Decreases Cytochrome C Release from Mitochondria after I/R injury and Increases Levels of the Anti-apoptotic Bcl-2 proteins.

<p>(<b>A</b>) Representative Western blot of cytosolic levels of GRK2 and cytochrome C (CytoC) in myocardial lysates purified from MCM, GRK2fl/fl, GRK2iKO, and GRK2KO mice 30 min after I/R injury. (<b>B</b>) Mean±SEM of CytoC release in MCM and GRK2iKO mice under Sham conditions and post-I/R with n-sizes shown within the individual bars, *<i>p</i><0.05 MCM-I/R vs MCM-Sham, ^#<i>p</i><0.05 GRK2iKO-I/R vs. MCM-I/R mice. (<b>C</b>) Mean±SEM of CytoC release in GRK2fl/fl and GRK2KO mice under Sham conditions and post-I/R with n-sizes shown within the individual bars, *<i>p</i><0.05 GRK2fl/fl-I/R vs GRK2fl/fl-Sham, ^#<i>p</i><0.05 GRK2KO-I/R vs. GRK2fl/fl-I/R mice. (<b>D</b>) Representative Western blot showing GRK2, Bcl-2 and Bcl-xl protein levels in myocardial lysates purified from Sham or post-I/R GRK2fl/fl control mice or constitutive GRK2KO mice. (<b>E–F</b>) Quantitative levels (Mean±SEM) of BCL-2 (<b>E</b>) or BCL-xL (<b>F</b>) found in myocardial lysates from Sham or post-I/R GRK2fl/fl control mice or GRK2KO mice, *<i>p</i><0.05 KO vs GRK2fl/fl (n = 4 per group). (<b>G</b>) Representative Western blot showing GRK2, Bcl-2 and Bcl-xl protein levels in myocardial lysates purified from Sham or post-I/R MCM control mice or inducible GRK2iKO mice. (H-I) Quantitative levels (Mean±SEM) of BCL-2 (<b>H</b>) or BCL-xL (<b>I</b>) found in myocardial lysates from Sham or post-I/R MCM control mice or GRK2iKO mice, *<i>p</i><0.05 KO vs GRK2fl/fl (n = 4 per group).</p

GRK2 Knock-down in Cultured Cardiomyocytes Reduces Cleaved Caspase 3 Levels and Increases Activated Akt and Bcl-2 Levels.

<p>(<b>A</b>) Representative Western blot for GRK2, total Akt, phospho-Akt (as in Fig. 7), Bcl-2, BCl-xL, cleaved caspase-3 and GAPDH as a loading control cultured neonatal rat ventricular myocytes (NRVMs) following 3 day treatment with control or GRK2-specific siRNA (see Methods) under basal conditions or after a challenge with Chelerythrine (Chele, 10 µM). (<b>B</b>) Quantitative assessment of GRK2 levels in NRVMs 3 days after siRNA treatment (control or GRK2 specific siRNA), *<i>p</i><0.05 GRK2-siRNA vs. Control-siRNA (ANOVA, n = 4 per group). (<b>C–F</b>) Quantitative assessment of protein levels of phosphor-Akt normalized to total Akt (<b>C</b>); Bcl-2 (<b>D</b>); Bcl-xl (<b>E</b>); and cleaved caspase-3 (<b>F</b>) from the corresponding NRVMs, *<i>p</i><0.05 between groups as indicated in each graph, n = 4.</p

Cardiac GRK2 KO Mice have Reduced Post-I/R Myocardial Apoptosis.

<p>(<b>A</b>) Representative photomicrographs of in situ detection of DNA fragments (TUNEL) in myocardial sections from Sham or post-I/R mice from the listed groups (MCM and GRK2fl/fl as controls as GRK2iKO and GRK2KO). Total nuclei were labeled with DAPI (blue), and apoptotic nuclei were detected by TUNEL staining (green) and a-actin labeled myocytes in red. (<b>B,C</b>) Mean±SEM of the percentage of TUNEL positive nuclei per total cell number per field (<b>B</b>) and percentage of TUNEL positive myocytes per field (<b>C</b>), *<i>p</i><0.05 vs. MCM and GRK2fl/fl control groups (ANOVA). (<b>D–F</b>) Activity (mean±SEM) of caspase-3 (<b>D</b>), -8 (<b>E</b>), and -9 (<b>F</b>) in all groups with n-sizes per group listed at bottom of graphs, *<i>p</i><0.05 vs. MCM and GRK2fl/fl control groups.</p

Loss of Cardiac GRK2 Expression Improves Post-I/R Hemodynamic Function.

<p>(<b>A</b>) Representative original LV dP/dt data tracing acquired from a Sham animal. Data was recorded at baseline (B) and upon isoproterenol treatment (0.1–1.0 ng/mouse). (<b>B–E</b>) Hemodynamic data (mean±SEM) recorded B and after cumulative doses of isoproterenol administration in Sham control mice (n = 8) and post-I/R groups (MCM, n = 8, GRK2fl/fl, n = 6, GRK2iKO, n = 9, and GRK2KO, n = 6) at 24 hrs after I/R. Measurements include LV +dP/dt_max (<b>B</b>); LV -dP/dt_min (<b>C</b>); LV end diastolic pressure (LVEDP (<b>D</b>); and heart rate (HR) (<b>E</b>). *<i>p</i><0.05 GRK2iKO or GRK2 KO vs. MCM and GRK2fl/fl control groups(two-way ANOVA).</p

Cardiac GRK2 Expression in Knockout Mice.

<p>(<b>A</b>) Whole heart GRK2 expression from MerCreMer (MCM), GRK2fl/fl (GRK2f/f) and GRK2iKO (inducible KO) groups before and after the treatment with tamoxifen. * <i>p</i><0.05 vs. GRK2f/f, MCM and GRK2iKO treated with vehicle groups. (<b>B</b>) GRK2 expression in cardiomyocytes isolated from GRK2iKO and GRK2f/f control mice. ** <i>p</i><0.05 vs. GRK2f/f. (<b>C</b>) Whole heart GRK2 expression from constitutive GRK2KO mice and GRK2fl/fl control mice. ** <i>p</i><0.01 vs. GRK2f/f.</p

Activated Akt is Increased in Post-I/R GRK2 KO Hearts.

<p>(<b>A</b>) Representative Western blots for GRK2, total Akt and phospho-Akt (activated via phosphorylation of Ser 473) in myocardial lysates of Sham and post-I/R (30 min I and 15 min R) GRK2fl/fl control mice and constitutive GRK2KO mice. (<b>B</b>) Quantitative levels (mean±SEM) of phospho-Akt normalized to total Akt found in myocardial lysates from Sham or post-I/R GRK2fl/fl or GRK2KO mice, *<i>p</i><0.05 KO vs GRK2fl/fl (n = 4 per group), ^#<i>p</i><0.05, GRK2fl/fl-I/R vs. GRK2fl/fl-Sham, **<i>p</i><0.01, GRK2KO-I/R vs. GRK2fl/fl-I/R and Sham groups (ANOVA, n = 4 pre group). (<b>C</b>) Representative Western blots for GRK2, total Akt and phospho-Akt in myocardial lysates of Sham and post-I/R MCM control mice and inducible GRK2KO mice (as in A-B). (<b>D</b>) Quantitative levels (mean±SEM) of phospho-Akt normalized to total Akt found in myocardial lysates from Sham or post-I/R MCM or GRK2iKO mice, **<i>p</i><0.01, MCM-IR vs. MCM sham; ^##<i>p</i><0.01, GRK2iKO-I/R vs. MCM-I/R and Sham groups (ANOVA, n = 4 pre group).</p

Cardiac GRK2 KO Mice have Reduced Post-I/R Infarct Size.

<p>(A) Representative photographs of TTC-stained mouse heart sections obtained from listed groups 24 hrs after a Sham procedure of I/R injury. (<b>B</b>) Graphic representation of the LV infarct size expressed as percentage of total ischemic area (Area at risk, AAR) in each group (S, n = 3, MCM, n = 8, GRK2fl/fl, n = 9, GRK2iKO, n = 15, and GRK2KO, n = 8). *<i>p</i><0.05, vs. MCM and GRK2fl/fl control mice (ANOVA). (<b>C</b>) Percentage of LV AAR for each group.</p

Loss of Cardiac GRK2 Expression Improves Post-I/R Cardiac Function.

<p>(<b>A</b>) Representative M-mode echocardiography recordings from Sham control mice (S, n = 8) and 24 hr post-I/R mice from different groups (MCM, n = 10, GRK2fl/fl, n = 7, GRK2iKO, n = 14, and GRK2KO, n = 9). (<b>B-E</b>) Mean±SEM of measured values from these groups of mice for LV internal diastolic dimension (LVIDd), (<b>B</b>); D, LV ejection fraction (LV EF%), (<b>C</b>); LV fractional shortening (LV FS%), (<b>D</b>); and heart rate (HR), (<b>E</b>). * <i>p</i><0.05 vs. MerCreMer (MCM) or GRK2fl/fl control groups.</p

CaMKII/ERK-dependent regulation of the hypertrophy marker ANF.

<p><b>A:</b> H9C2 cells at ≈ 70% confluence were incubated 1 h at 37°C with 5 mL DMEM containing purified adenovirus at a multiplicity of infection (moi) of 100:1, encoding either the kinase-dead (CaMKII-DN, rCaMKIIdelta, K42M), or the wild type (CaMKII-WT, rCaMKIIdelta) variant of CaMKII or the empty virus as a negative control (Ctr). 48 h after the infection, the cells were stimulated with PE 100 nM for 24 h. Total RNA was isolated from H9C2s using TRIzol reagent, and cDNA was synthesized by means of a Thermo-Script RT-PCR System, following the manufacturer’s instruction. Then ANP gene expression was evaluated by real-time PCR. Results are expressed as mean±SEM from 3 independent experiments. The ratio of fold change was calculated using the Pfaffl method[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130477#pone.0130477.ref036" target="_blank">36</a>]. * = p<0.05 vs Ctrl; # = p<0.05 vs PE. <b>B:</b> The H9C2s infected with adenoviruses encoding wilde type CaMKII (CaMKII-WT) and kinase dead CaMKII (CaMKII-DN) were stimulated with PE 100 nM for 24 h. Total cell lisates were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were corrected by Actin densitometry. Data from the immunoblots were quantified by densitometric analysis.* = p<0.05 vs Ctrl. Each data point in all graphs represents the mean±SEM of 3 independent experiments. <b>C:</b> H9C2 cells were pretreated with CaMK inhibitor KN93 (5 μmol/L), the selective inhibitors AntCaNtide (10 μmol/L) and tat-CN17β (10 μmol/L) and ERK specific inhibitor pathway UO126 (10 μmol/L) for 30 min. and then stimulated with PE (100 nmol/L) for 24 h. cDNA was synthesized from RNA obtained from H9C2s as indicated above. The ANF gene expression was evaluated by real-time PCR. Results are expressed as mean±SEM from 3 independent experiments. The ratio of fold change was calculated using the Pfaffl method[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130477#pone.0130477.ref036" target="_blank">36</a>]. * = p<0.05 vs Ctrl; # = p<0.05 vs PE.</p

The effect of CaMKII selective peptide inhibitors in vivo in SHRs.

<p><b>A:</b> SHRs were subjected to intramural cardiac injections at the age of 13 week and repeated after 7 and 14 days. Transthoracic echocardiography was performed at each drug administration and once more at the day of euthanasia in untreated WKY rats, AntCaNtide SHRs, tat-CN17β SHRs, and control SHRs (SHR) using a dedicated small-animal high-resolution-ultrasound system (Vevo 770, VisualSonics). ANTCaNtide-SHRs and tat-CN17β-SHRs caused reduction of interventricularseptal (IVS) thickness compared to SHR (*<i>P</i><0.05 vs WKY; #<i>P</i><0.05 vs SHR). <b>B, C:</b> At the end of treatment, rats were weighed and then euthanized. Hearts were immediately removed, rinsed 3 times in cold PBS and blotted dry, weighed and then rapidly frozen. The HW/BW ratio (<b>B</b>) and LVM/BW ratio (<b>C</b>) were measured in ANTCaNtide-SHRs and tat-CN17β-SHRs and compared to WKY and SHR hearts (*<i>P</i><0.05 vs WKY; #<i>P</i><0.05 vs SHR). <b>D:</b> Cardiac Performance at the end of the treatment was assessed by ultrasound. All measurements were averaged on 5 consecutive cardiac cycles and analysed by two experienced investigators blinded to treatment (GS, MC). No differences were observed in LVFS and LVEF in ANTCaNtide and tat-CN17β SHRs when compared with SHR (*P<0.05 vs WKY). <b>E:</b> To evaluate the involvement of BP values in CaMKII–dependent regulation of cardiac hypertrophy, SBP and DBP values were assessed in AntCaNtide- and tat-CN17β- treated and control. The pressure values ​​in the graph represent the measurements made at the end of treatment (*<i>P</i><0.05 vs WKY-SBP; #<i>P</i><0.05 vs WKY-DBP). <b>F:</b> At the end of the treatment the total RNA was isolated from myocardial sample using TRIzol reagent, and cDNA was synthesized by means of a Thermo-Script RT-PCR System, following the manufacturer’s instruction. ANP gene expression was evaluated by real-time PCR in AntCaNtide- and tat-CN17β-SHR rats compared with WKY and SHR (*<i>P</i><0.05 vs WKY). Results are expressed as mean±SEM from 3 independent experiments. The ratio of fold change was calculated using the Pfaffl method[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130477#pone.0130477.ref036" target="_blank">36</a>].</p