2013 Pharmacogenomics mTOR.pdf

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px Times; min-height: 14.0px} p.p2 {margin: 6.0px 0.0px 0.0px 0.0px; text-align: justify; line-height: 10.1px; font: 10.0px Times; color: #2c2728} span.s1 {font: 12.0px Times; color: #000000} <p>The mTOR signaling pathway integrates inputs from a variety of upstream stimuli to regulate diverse cellular processes including proliferation, growth, survival, motility, autophagy, protein synthesis and metabolism. The mTOR pathway is dysregulated in a number of human pathologies including cancer, diabetes, obesity, autoimmune disorders, neurological disease and aging. Ongoing clinical trials testing mTOR-targeted treatments number in the hundreds and underscore its therapeutic potential. To date mTOR inhibitors are clinically approved to prevent organ rejection, to inhibit restenosis after angioplasty, and to treat several advanced cancers. In this review we discuss the continuously evolving field of mTOR pharmacogenomics, as well as highlight the emerging efforts in identifying diagnostic and prognostic markers, including miRNAs, in order to assess successful therapeutic responses. <br></p

2015 PNAS HF Santulli (First and Corresponding Author).pdf

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 8.0px Helvetica} span.s1 {font: 5.5px Helvetica} <p>Calcium (Ca2+) released from the sarcoplasmic reticulum (SR) is crucial</p> <p>for excitation–contraction (E–C) coupling. Mitochondria, the major</p> <p>source of energy, in the form of ATP, required for cardiac contractility,</p> <p>are closely interconnected with the SR, and Ca2+ is essential for optimal</p> <p>function of these organelles. However, Ca2+ accumulation can</p> <p>impair mitochondrial function, leading to reduced ATP production</p> <p>and increased release of reactive oxygen species (ROS). Oxidative</p> <p>stress contributes to heart failure (HF), but whether mitochondrial</p> <p>Ca2+ plays a mechanistic role in HF remains unresolved. Here, we</p> <p>show for the first time, to our knowledge, that diastolic SR Ca2+</p> <p>leak causes mitochondrial Ca2+ overload and dysfunction in a murine</p> <p>model of postmyocardial infarction HF. There are two forms of Ca2+</p> <p>release channels on cardiac SR: type 2 ryanodine receptors (RyR2s)</p> <p>and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s). Using murine</p> <p>models harboring RyR2 mutations that either cause or inhibit SR</p> <p>Ca2+ leak, we found that leaky RyR2 channels result in mitochondrial</p> <p>Ca2+ overload, dysmorphology, and malfunction. In contrast, cardiacspecific</p> <p>deletion of IP3R2 had no major effect on mitochondrial fitness</p> <p>in HF. Moreover, genetic enhancement of mitochondrial antioxidant</p> <p>activity improved mitochondrial function and reduced posttranslational</p> <p>modifications of RyR2 macromolecular complex. Our data demonstrate</p> <p>that leaky RyR2, but not IP3R2, channels cause mitochondrial</p> <p>Ca2+ overload and dysfunction in HF.</p

PKA inhibition does not modify PE-induced CaMKII/ERK activation in H9C2 cardiomyoblasts.

<p><b>A:</b> H9C2 cardiomyoblasts were transfected with PKA-I and then stimulated with 100 nm phenylephrine (PE) for 15 min. Total cell extracts were analyzed by western blot for phosphothreonine 286 CaMKII (pCaMKII) and CaMKII with specific antibodies. pCaMKII levels were corrected by CaMKII densitometry. *, <i>P</i> < 0.05 <i>vs</i>. Ctrl. <b>B:</b> H9C2s were stimulated with PE after transfection with PKA-I. Total lysates were analysed by WB for pERK with specific antibody. pERK1/2 levels were corrected by ERK1/2 densitometry. * = p<0.05 vs Ctrl. <b>C:</b> To evaluate that PKA inhibition does not modify CaMKII/ERK interaction, H9C2s were stimulated with 100 nmol/L PE for 15 minutes following 30 min. pretreatment with PKA-I (10 μmol/L). CaMKII was immunoprecipitated from cell lysates using a specific anti- CaMKII antibody, and ERK1/2 was visualized by WB to evaluate its association with CaMKII. ERK1/2 levels were corrected by CaMKII densitometry. * = p<0.05 vs Ctrl. <b>D:</b> To confirm the interaction between CaMKII and ERK in H9C2s, total cell lysates were immunoprecipitated using anti-ERK1/2 antibody, and subjected to western blot using anti-CAMKII antibody. CaMKII levels were corrected by ERK1/2 densitometry. * = p<0.05 vs Ctrl. <b>E:</b> H9C2s were stimulated with PE after trasfection with PKA-I. Subsequently, nuclear extract were prepared from the cells as indicated in the methods section. Nuclear extracts were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were averaged and normalized to histone 3 densitometry. *, <b><i>P</i></b> < 0.05 <b><i>vs</i>.</b> Ctrl. <b>F:</b> To confirm that PE induced ERK nuclear localization was independent from PKA inhibition, H9C2s were transfected with PKA-I and then stimulated with PE. Nuclear extracts were analyzed by WB for total ERK1/2 with specific antibody. ERK1/2 levels were normalized to histone 3 densitometry. *, <b><i>P</i></b> < 0.05 <b><i>vs</i>.</b> Ctrl. Data from all immunoblots were quantified by densitometric analysis. Each data point in all graphs represents the mean±SEM of 3 independent experiments.</p

CaMKII selective peptide inhibitors reduces myocardial fibrosis in SHRs.

<p><b>A:</b> Paraffin-blocked tissues from left ventricle were sectioned and stained with Masson's trichrome after three weeks of CaMKII selective inhibitors (×20). SHR treated with AntCaNtide and tat-CN17β showed a decrease of interstitial fibrosis and reduced cardiomyocytes size when compared to SHR untreated group. The section stained with Masson's trichrome of WKY was used as control. <b>B:</b> Cardiomyocytes size was measured by Image J software, and means of areas are showed in the histogram ((*P<0.05 vs WKY; # P<0.05 vs SHR). Images are representative of 3 independent experiments (magnification ×60; black bar = 100 μm). <b>C:</b> Quantification of fibrosis was done by Image J software, and percent of fibrotic areas compared to WKY are shown in the histogram (*P<0.05 vs WKY). Images are representative of 3 independent experiments. <b>D, E:</b> To confirm that intramyocardial injection with CAMKII inhibitors AntCaNtide and tat-CN17β blunted interstitial fibrosis we have evaluated mRNA levels of fibrosis biomarkers such as collagen type I (<b>D</b>) and collagen type III (<b>E</b>) using RT-PCR. *<i>P</i><0.05 vs WKY; # <i>P</i><0.05 vs SHR. Results are the mean of 3 independent experiments.</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

Inhibition of ERK pathway downregulates PE-induced CaMKII activation in H9C2 cardiomyoblasts.

<p><b>A:</b> H9C2 cardiomyoblasts were exposed to UO126 (10 μmol/L) for 30 min., and then stimulated with 100 nM phenylephrine (PE) for 15 min. Total cell extracts were analyzed by western blot for phosphothreonine 286 CaMKII (pCaMKII) and CaMKII with specific antibodies. pCaMKII levels were corrected by CaMKII densitometry. *, <i>P</i> < 0.05 <i>vs</i>. Ctrl; #, <i>P</i> < 0.05 <i>vs</i>. PE. <b>B:</b> H9C2s were stimulated with PE after pretreatment with UO126 (10 μmol/L for 30 min.). Total lisates were analyzed by WB for pERK with specific antibody. pERK1/2 levels were corrected by ERK1/2 densitometry. * = p<0.05 vs Ctrl; # = p<0.05 vs PE. <b>C:</b> H9C2s were stimulated with 100 nmol/L PE for 15 minutes following 30 min. pretreatment with UO 126 (10 μmol/L). CaMKII was immunoprecipitated from cell lysates using a specific anti- CaMKII antibody, and ERK (ERK1/2) was visualized by WB to evaluate its association with CaMKII. ERK1/2 levels were corrected by CaMKII densitometry. * = p<0.05 vs Ctrl;# = p<0.05 vs PE. <b>D:</b> To confirm the interaction between CaMKII and ERK in H9C2s, total cell lysates were immunoprecipitated using anti-ERK1/2 antibody, and subjected to western blot using anti-CAMKII antibody. CaMKII levels were corrected by ERK1/2 densitometry.* = p<0.05 vs Ctrl; # = p<0.05 vs PE. <b>E:</b> After pharmacological inhibition of ERK and stimulation with PE, nuclear extract from H9C2s were prepared as indicated in the methods section. Nuclear extracts were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were averaged and normalized to histone 3 densitometry. *, <b><i>P</i></b> < 0.05 <b><i>vs</i>.</b> Ctrl; # = p<0.05 vs PE. <b>F:</b> To confirm the effects PE induced ERK nuclear localization after pretreatment with UO126, nuclear extracts were analyzed by WB for total ERK1/2 with specific antibody. ERK1/2 levels were normalized to histone 3 densitometry. *, <b><i>P</i></b> < 0.05 <b><i>vs</i>.</b> Ctrl; # = p<0.05 vs PE. Data from all immunoblots were quantified by densitometric analysis. Each data point in all graphs represents the mean±SEM of 3 independent experiments.</p

Effects of CaMKII inhibition on CaMKII/ERK pathway <i>in vivo</i> in SHR.

<p><b>A</b>: After three weeks of treatments, heart were harvested, weighted, and samples from WKY, SHR- AntCaNtide, SHR- tat-CN17β, and SHR-Control total lysates were prepared from the left ventricular samples. Whole lysates were subjected western blotting analysis with anti‐CAMKII antibody. CAMKII levels were corrected by Actin densitometry.* = p<0.05 vs WKY. <b>B</b>: To assess CAMKII phosphorylation levels in LV after CaMKII inhibitors pretreatment, total lysate samples from WKY, SHR-AntCaNtide, SHR- tat-CN17β, and SHR-Control were analyzed by WB for anti-phosphothreonine 286 CaMKII antibody (pCaMKII). pCaMKII levels were corrected by Actin densitometry.* = p<0.01 vs WKY. <b>C:</b> Total cell extracts of LV from WKY, SHR-AntCaNtide, SHR- tat-CN17β, and SHR-Control were analyzed by WB with anti-pERK (pERK1/2) or anti- total ERK1/2. pERK1/2 levels were corrected by total ERK1/2 densitometry. * = p<0.01 vs WKY. <b>D:</b> To evaluate the effects of CaMKII inhibition on CaMKII subcellular compartmentalization, nuclear extract from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β were prepared as indicated in methods. Nuclear extracts were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were averaged and normalized to histone 3 densitometry.*, <i>P</i> < 0.05 <i>vs</i>. WKY. <b>E:</b> The nuclear extract from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β were analyzed by WB for total ERK with specific antibody to test the effects of CaMKII inhibitors on ERK subcellular compartmentalization. CaMKII levels were averaged and normalized to histone 3 densitometry.*, <i>P</i> < 0.05 <i>vs</i>. WKY. <b>F</b>: To examine the association between CaMKII and ERK in the left ventricle from SHR following intramyocardial injections, total cell lysate from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β, was immunoprecipitated using anti-CaMKII antibody and subjected to WB with anti-ERK antibody. ERK levels were averaged and normalized to IgG densitometry. *<i>P</i> < 0.05 vs. WKY. Data from all immunoblots presented here were quantified by densitometric analysis. Each data point in all graphs represent the mean±SEM of 3 independent experiments.</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

CaMKII isoforms.

<p><b>A:</b> To evaluate the expression of CaMKII isoforms in ventricular adult cardiomyocytes from WKY rats, the total cell lysates were immunoprecipitated using anti-CaMKII antibody (lane a) or anti-CaMKII α, β γ or δ specific antibodies (lane b) and together to total samples from WKY cardiomyocytes (lane d) and H9C2 cardiomyoblasts (lane e) were analyzed by western blot with anti-CaMKII α, β γ or δ antibodies as indicated. Total extracts from rat brain (lane f), mouse brain (lane g) and mouse heart (lane h) were used as controls. Total lysates from WKY cardiomyocytes with specific antibody without A/G agarose beads were used as negative control (lane c). <b>B:</b> Total RNA from H9C2 cell line and isolated ventricular cardiomyocytes was extracted with standard methods. RT-PCR for CaMKII α, β, γ, and δ was performed as indicated in methods. The representative graph indicates the relative amounts of transcripts for CaMKII isoforms in H9C2s and ventricular adult myocytes from WKY rats. Cycle threshold (Ct) values from 3 independent experiments were normalized to the internal β-actin control. The ratio of fold change was calculated using the Pfaffl method. * = p<0.05 vs CaMKIIα; # = p<0.05 vs CaMKIIβ.</p