Decrease In EF Research Articles

Abstract 442: Cardiomyocyte Specific mTORC1 Deletion Precipitates Interstitial Fibrosis, Heart Failure And Loss Of Female Cardio Protection

Mechanistic target of rapamycin (mTOR) kinase operates in two functionally and structurally distinct multiprotein complexes mTORC1 and mTORC2. mTORC1 including its defining raptor protein is a critical mediator of myocardial growth. We induced cardiomyocyte specific raptor-KO (cKO) deletion to investigate a sex specific role of mTORC1 during cardiac adaptation in normotensive DOCA-salt mice. Untypical for this mild cardiac stress model, raptor-cKO females had sharper decrease in EF (51 vs. 26%) than males (50 vs. 32%) already at 3 weeks after start of DOCA-salt challenge. Male raptor-cKO mice mounted eccentric hypertrophic response upon DOCA-salt indicated by heart weight/tibia length ratios (6.7 to 8.0) as compared to their WT litters (6.6 to 7.6). In contrast female raptor-cKO (5.8 to 6.4) minimally differed from their litters (5.8 to 6.6). Severe dilative phenotype was detected in both sexes regardless of DOCA-salt challenge. In addition, we found tremendous diffuse left ventricular fibrosis which was also more prominent in raptor-cKO females. Collagen depositions in the LV visualized by Sirius red staining increased 10fold in males (1.0 to 10.6%) and 15fold in females (0.9 to 15.7%), respectively. Expression of raptor protein in the heart was diminished by 80% in raptor cKO mice of both sexes what lead to similar extent of decreased activity of direct mTORC1 downstream targets phospho-S6 ribosomal protein and phospho-4E-BP1Ser65 (68% reduction in males vs. 61% in females. Over-compensatory mTORC2 activation in cKO animals via phospho-AKTSer473 (12fold increase) and induction of anti-apoptotic signaling was similar in both sexes. However, raptor-cKO females failed to up-regulate expression of mitochondrial genes such as Mtnd1 (ND1), mt-Co1 (COX1) and Atp5i (ATP5k), stressing sex differences in mTORC1 dependent mitochondrial adaptation. In addition, transmission electron microscopy (TEM) points towards structural alterations of mitochondrial morphology. Intact mTORC1 function is important for myocardial adaptation in both sexes. More severe phenotype in females further implicates mTORC1 to be essential for intrinsic female cardio protection.

Mutation of Kvβ2 active site attenuates cardiac injury: role of repolarization reserve (864.2)

Cardiac Kvβ2 subunit alters gating and expression of potassium channels. However, the physiological significance remains unknown. Therefore, we evaluated the role of Kvβ2 in cardiac injury. We used 14 week male wild type (Wt) or mutant Kvβ2:PM (active site mutant; Y90F) mice, infused with saline or isoproterenol (Iso, 30mg/kg; bw/day) for 14 days. Iso treatment resulted in cardiac hypertrophy in both mice as noted by histomorphological analysis. Echocardiographic assessment revealed significant decrease (p <0.05) in EF% and FS% upon Iso stress in both Wt (71.1±1.7 vs. 55.9±1.7; 40±1.4 vs. 29.0±1.2) and PM (66.3±2.7 vs. 57 ± 2.8; 36.2 ± 2.0 vs. 30.0 ± 1.9), mice. Ex vivo recording of action potential durations (APD) showed repolarization depletion in Wt saline vs. Iso infused mouse hearts at APD60 (11.9±0.7 vs. 31.6±1.1) and APD80, 43.1 ± 1.9 vs. 51.9 ± 1.9), respectively. However, in PM mice Iso did not induce APD prolongation (APD60, 19.7±1.1 vs. 24.2±1.8 or APD80, 49.8±1.8 vs. 49.3±2.0) suggesting protection. Similarly, ECG analysis showed QTc interval (59.7±2.4 vs. 68.6±2.0, ms) change in Wt saline vs. Wt Iso groups, which is not observed in PM mice (70±1.7 vs. 68.9±0.6). Expression of key ion channels (Kv4.2, Kv1.4 and Kv1.5) was unaltered in PM mice, but significantly depleted in Wt with Iso treatment. Together, Kvβ2 active site mutant restores repolarization reserves and offers protection against cardiac injury.Grant Funding Source: HL102171

First-principles study on the phase transition, elastic properties and electronic structure of Pt3Al alloys under high pressure

The phase transition, formation enthalpies, elastic properties and electronic structure of Pt3Al alloys are studied using first-principle approach. The calculated results show that the pressure leads to phase transition from tetragonal structure to cubic structure at 60GPa. With increasing pressure, the elastic constants, bulk modulus and shear modulus of these Pt3Al alloys increase linearly and the bond lengths of Pt–Al metallic bonds and the peak at EF decrease. The cubic Pt3Al alloy has excellent resistance to volume deformation under high pressure. We suggest that the phase transition is derived from the hybridization between Pt and Al atoms for cubic structure is stronger than that of tetragonal structure and forms the strong Pt–Al metallic bonds under high pressure.

Effect of formula of removing both phlegm and blood stasis in improving cardiac function of Chinese mini-swine with coronary heart disease of phlegm-stasis cementation syndrome

To evaluate that the effect of formula of removing both phlegm and blood stasis in improving cardiac function of Chinese mini-swine with coronary heart disease of phlegm-stasis cementation syndrome. Totally 36 Chinese mini-swine were randomly divided to six groups: the normal control group, the model group, the Danlou tablet group, and Tanyu Tonzhi Fang(TYTZ) groups with doses of 2. 0, 1. 0 and 0. 5 g kg-1, with six in each group. Except for the normal control group, all of other groups were fed with high-fat diet for 2 weeks. Interventional balloons are adopted to injure their left anterior descending artery endothelium. After the operation, they were fed with high-fat diet for 8 weeks to prepare the coronary heart disease model of phlegm-stasis cementation syndrome. After the operation, they were administered with drugs for 8 weeks. The changes in the myocardial ischemia were observed. The changes in the cardiac function and structure were detected by cardiac ultrasound and noninvasive hemodynamic method. Compared with the normal control group, the model group showed significant increase in myocardial ischemia and SVR and obvious decrease in CO, SV and LCW in noninvasive hemodynamic parameters (P <0.05 or P <0.01). The ultrasonic cardiogram indicated notable decrease in IVSd, LVPWs, EF and FS, and remarkable increase in LVIDs (P<0. 05 orP<0.01). Compared with the model group, TYTZ could reduce the myocardial ischemia, strengthen cardiac function, and improve the abnormal cardiac structure and function induced by ischemia (P <0. 05 or P <0. 01). TYTZ shows a significant effect in improving cardiac function of Chinese mini-swine with coronary heart disease of phlegm-stasis cementation syndrome. The clinical cardiac function detection method could be adopted to correctly evaluate the changes in the post-myocardial ischemia cardiac function, and narrow the gap between clinical application and basic experimental studies.

Resting Heart Rate as Predictor for Left Ventricular Dysfunction and Heart Failure: MESA (Multi-Ethnic Study of Atherosclerosis)

The objective of this study was to investigate the relationship between baseline resting heart rate and incidence of heart failure (HF) and global and regional left ventricular (LV) dysfunction. The association of resting heart rate to HF and LV function has not been well described in an asymptomatic multi-ethnic population. Resting heart rate was measured in participants in the MESA (Multi-Ethnic Study of Atherosclerosis) trial at inclusion. Incident HF was registered (n = 176) during follow-up (median 7 years) in those who underwent cardiac magnetic resonance imaging (n = 5,000). Changes in ejection fraction (ΔEF) and peak circumferential strain (Δεcc) were measured as markers of developing global and regional LV dysfunction in 1,056 participants imaged at baseline and 5 years later. Time to HF (Cox model) and Δεcc and ΔEF (multiple linear regression models) were adjusted for demographics, traditional cardiovascular risk factors, calcium score, LV end-diastolic volume, and mass in addition to resting heart rate. Cox analysis demonstrated that for 1 beat/min increase in resting heart rate, there was a 4% greater adjusted relative risk for incident HF (hazard ratio: 1.04; 95% CI: 1.02 to 1.06; p < 0.001). Adjusted multiple regression models demonstrated that resting heart rate was positively associated with deteriorating εcc and decrease in EF, even when all coronary heart disease events were excluded from the model. Elevated resting heart rate was associated with increased risk for incident HF in asymptomatic participants in the MESA trial. Higher heart rate was related to development of regional and global LV dysfunction independent of subclinical atherosclerosis and coronary heart disease. (Multi-Ethnic Study of Atherosclerosis [MESA]; NCT00005487).

Unsuccessful treatment of accelerated hypertension and persistent hyperkinetic state in a haemodialysed patient with high-output arteriovenous fistula

The case we describe concerns a rapid progression of heart failure (HF) in a patient with hypertension crises (HC), haemodialysed (HD) with the use of a high-output arteriovenous fistula (AVF). The 48-year-old male with a body mass index of 19.27 kg/m2, renal insufficiency due to glomerulonephritis, treated with HD for two years (3 × 4.0 h/week), and with uncontrolled hypertension was admitted to the Military Institute of Medicine. On examination, reduced exercise tolerance, paroxysmal nocturnal dyspnoea and blood pressure (BP) 190/90 mm Hg were found. Laboratory tests revealed NT-pro B-type natriuretic peptide 302,009.0 pg/mL (n < 300.0). Echocardiography (ECHO) showed left ventricular (LV) hypertrophy (LV mass index [LVMI] 345.81 g/m2), enlarged LV diastolic diameter [LVDD] 7.15 cm), with slightly decreased ejection fraction (EF 49%), Doppler signs of elevated cardiac output (CO 15.05 L/min; cardiac index (CI) 8.91 L/min/m2), and pseudonormalised pattern of mitral inflow with elevated LV diastolic pressure (E/E’ 31). In ultrasonographic examination of left shoulder AVF, an aneurysmal expansion of the lumen from 5.5 mm to 35 mm (Fig. 1), and significantly increased flow up to 2,532 mL/min (Fig. 2) were detected. Cardiopulmonary recirculation rate (CPR) was 0.17. Computed tomography excluded renal artery stenosis. In polysomnography, heavy central apnoea with an apnoea/hypopnoea index of 35.4/h was found. In 48-h ambulatory BP monitoring (ABPM), BP was 162/95 mm Hg. Treatment of sleep apnoea was implemented by a continuous positive airway pressure device, but this did not improve BP control. Upon discharge from hospital, the patient was overtreated with eight hypotensive drugs. After a month, HC occurred (BP 220/120 mm Hg) associated with acute coronary syndrome without ST-segment elevation. Coronarography found non-obstructive atherosclerotic plaques. Because of the suspicion of hyperkinetic state related to a lack of BP control, AVF was narrowed by implanting a polytetrafluorethylene graft of 4 mm diameter (Fig. 3). Despite AVF flow being reduced to 1,400 mL/min, after two months another HC (BP 220/125 mm Hg) with pulmonary oedema was presented. The next HC took place three months later. Although ECHO showed features of LV hypertrophy regression (LVMI 335.62 g/m2), and significant decreases of CO (8.16 L/min) and CI (4.86 L/min/m2), a decrease of EF (40%) and an LV dilatation enlargement (LVDD 7.7 cm) were also observed. AVF flow increased up to 1,750 mL/min and significant CPR 0.21 was indicated. In 24-h ABPM, BP was 173/105 mm Hg. The hypotensive treatment used has been confirmed by experts and seems to be optimal and safe for the cardiovascular system. Probably, AVF banding was performed too late, and progression of HF and lack of BP control improvement were observed. In hyperkinetic AVF, blood flow exceeds 2,000 mL/min and CPR is over 0.2–0.3. Currently, in cases of difficulties in surgical AVF creation and the necessity of haemodialysotherapy termination as a result of a lack of vascular access, banding or closure of the AVF is a very rare event. However, in some cases, early ligation of AVF may be the only effective procedure, especially when there is rapid progression of HF.

Impact of pulmonary vein antrum isolation on left atrial size and function in patients with atrial fibrillation

The success of PVAI in eliminating AF has been proven; however, its impact on the LA remains uncertain. This study aimed to determine the impact of pulmonary vein antrum isolation (PVAI) on left atrial (LA) size and function in patients with atrial fibrillation (AF). Consecutive patients with AF were included (n = 206). Magnetic resonance imaging (MRI) was performed before and after PVAI in all patients. A subgroup (n = 52) underwent delayed enhancement MRI. Maximal LA volume (LAVmax) and minimal LA volume (LAVmin) were assessed by Simpson's rule. LA function was determined by calculating LA ejection fraction (LA EF). LA fibrosis was manually encircled and summed in the region of interest. Single procedure success rate was 64 %. LAVmax decreased post-ablation in all patients (125.1 to 111.9 ml, p < 0.001). LAVmin only decreased in patients with a successful outcome post-ablation (65.6 to 58.8 ml, p < 0.001). As a result, LA EF only showed a marked reduction in patients with AF recurrences (42.7 % to 37.9 %, p < 0.001). Post-ablation LA fibrosis could be visualized in 77 % of patients who underwent delayed enhancement MRI (mean amount 1.4 cm(3)). LA fibrosis showed no correlation with the decrease in LAVmax or LA EF. PVAI resulted in a reduction of LAVmax in all patients, indicating an effect of ablation induced fibrosis. LAVmin only decreased in patients with a successful outcome, indicating an effect of reverse atrial remodeling. As a result, LA function post-ablation was preserved in patients with a successful outcome and decreased in patients with AF recurrence.

2013 Late-Breaking Basic Science Abstracts

### 21998 #### Enhanced Myocardial Repair with CardioChimeras Pearl J Quijada, Jonathan D Cubillo, Claudio Staub, Mark A Sussman; San Diego State Univ, San Diego, CA Dual stem cell transplantation of c-kit positive cardiac progenitor cells (CPCs) and mesenchymal stem cells (MSCs) after infarction support modest improvements in cardiac function, however there are currently no reports substantiating a single stem cell type supporting both direct and indirect mechanisms of myocardial repair. Therefore we created a CardioChimera, a stem/progenitor cell formed by fusion between CPCs and MSCs, resulting in a unique progeny superior to either individual precursor. CardioChimeras were purified after cell fusion with Hemagglutinating virus of Japan and expanded clonally based on dual expression of fluorescent proteins mCherry and eGFP from CPCs and MSCs respectively. CardioChimeras are mono-nucleated, have comparable growth kinetics to parental CPCs and MSCs and display increased cellular size unrelated to cell cycle arrest and/or senescence. CardioChimeras have increased expression of cardiomyogenic lineage markers cardiac troponin T (2.5-fold), smooth muscle 22 (9.3-fold), and CD31 (10.7-fold) concomitantly associated with decreased c-kit protein expression (50%) relative to parent CPCs. Lineage commitment of CardioChimeras is bolstered by dexamethasone treatment measured by mRNA levels of cardiogenic genes and increases in active mitochondria (2.2-fold) after labeling with MitoTracker. Induction of apoptosis is blunted in cardiomyocytes co-cultured with CardioChimeras compared to co-culturing with CPCs and MSCs alone, or combination of non-fused parent cells. CardioChimeras enhance cardiomyocyte growth similar to parent MSCs owing to an increased propensity to secrete pro-growth factors. Collectively, CardioChimeras represent an adaptable cell therapy combining the beneficial properties of CPCs to undergo cardiac specific commitment as well as MSCs that foster an improved microenvironment with protective paracrine secretion. Clinically, CardioChimeras merge the application of distinct cell types into a single entity for increased engraftment, mitigation of inflammation and blunting the progression of heart failure by promoting myocardial regeneration. …

Abstract 18307: Intracoronary Infusion of Allogeneic Cardiosphere Derived Cells, During Acute Myocardial Infarction Delays but Eventually Does Not Limit Remodeling of the Left Ventricle

Objective: In animal models, it has been shown that myocardial cells regenerate even in the adult heart at very slow rates and that regeneration is stimulated after myocardial injury. However the endogenous mechanisms of regeneration are not adequate to limit the damage produced by an acute myocardial infarction.The objective of the study was to demonstrate if remodeling of the left ventricle is attenuated by intracoronary infusion of Allogeneic Cardiosphere Derived Cells (CDCs), at the ischemic phase of acute myocardial infarction, using a chronic ischemia- reperfusion model in swine. Method: In twelve pigs, AMI was induced by occlusion of Left Anterior Descending Coronary Artery for 60 minutes. At 60 minutes of ischemia, 6 animals received 5 million allogeneic CDCs (Group 1), and 6 (Group 2) normal saline, using a catheter that crossed the occlusion site. Five minutes after cell infusion, reperfusion was allowed. The follow up period was 30 days. All animals underwent transthoracic echocardiography before and immediately after the AMI, as well as at 15 and 30 days. Left ventricular end diastolic diameter (LVEDD), end-systolic diameter (LVESD) and ejection fraction (LVEF) were measured. Results: Group 1 animals did not demonstrate statistical significant increase in LV dimensions or decrease of EF at 15 days (LVEDD from 38mm to 42.6mm, p= 0.149, LVEF from 67.66% to 56.5%, p= 0.335). In contrast, Group 2 animals experienced significant decrease of LV function (LV EF from 56.5% to 48%, p=0.014) and a significant increase of LVESD (from 22.75mm to 28.5mm p=0.005) and LVEDD (from 32 mm to 37.75mm, p=0.009). However, animal in both groups had significant remodeling at 30 days There was an equivalent % reduction of LVEF (8.3% vs 7.6%, p=0.95) and increase of LVEDD and LVESD ( 6.5% vs 9.2 % p=0.3 and 6.3% vs 8.6% p=0.6 ,respectively) in Group 1 compared to Group 2. Conclusions: Infusion of allogeneic CDCs during acute AMI was safe and well tolerated hemodynamically, but produced only a transient benefit on LV function. This result may suggest that a re-infusion of cells is necessary at later time points after the index MI, in order to achieve sustained improvement.

Phase I Study Of The Combination Of Midostaurin, Bortezomib and Chemotherapy In Relapsed/Refractory Acute Myeloid Leukemia (AML): Targeting Aberrant Tyrosine Kinase Activity

BackgroundWe have shown that the proteasome inhibitor bortezomib is able to disrupt the physical interaction between the transcription factor Sp1 and NF-kB(p65), inhibiting Sp1 DNA-binding activity in myeloid blasts. (Liu, Blood 2008) We have also observed that both KIT and FLT3 promoters harbor Sp1 binding sites and that Sp1 induces transcription of these genes. We hypothesize that the Sp1/NF-kB pathway is a modulator of tyrosine kinase (TK) activity, and that administration of bortezomib with the tyrosine kinase inhibitor midostaurin will down regulate TKs at the transcription level and inhibit kinase activity in AML. The expectation is that two fold molecular targeting in combination with chemotherapy will result in a greater antileukemic effect. MethodsAdults with relapsed or refractory AML by WHO criteria and preserved organ function with ECOG ≤ 2 were eligible. Patients enrolled at dose levels (DL) 1 and 2 received midostaurin 50mg po BID d 1-14, bortezomib was dose escalated – DL 1: 1.0mg/m2 IV d 1,4,8,11; DL 2: 1.3mg/m2 IV d 1,4,8,11. Cycles were repeated every 28 d or until achievement of morphologic complete remission (CR) or CR with incomplete count recovery (CRi), for up to 3 cycles. Patients enrolled at DL 3 received mitoxantrone 4mg/m2, etoposide 40mg/m2, cytarabine 1g/m2 d1-6 (MEC) followed by midostaurin 50mg bid d 8-21 and bortezomib 1.3mg/m2 IV d 8,11,15,18. Due to the development of grade 3 peripheral neuropathy (PN) that was a dose limiting toxicity (DLT) in two patients treated at DL 3, bortezomib administration was changed from IV to subcutaneous (SQ) and the dose and schedule was changed to 1.0mg/m2 on d 8 and 15 on (DL 3A) - doses of midostaurin and MEC remained constant. Patients enrolled at DL 3 or 3A received one cycle of treatment, but were able to continue with maintenance bortezomib and midostaurin only for up to three cycles if they did not proceed to allogeneic stem cell transplantation (SCT). Responses were graded by International Working Group criteria for AML (Cheson, JCO 2013). Results11 patients enrolled to DL 1 and 2; 7 had relapsed disease (5 with CR1< 1 year) and 4 were primary refractory. The median age was 67 (range, 64-76) and 4 patients were FLT3-ITD or FLT3-TKD positive. According to European LeukemiaNet (ELN) genetic risk groups, 6, 3, and 2 patients had intermediate I, intermediate II and adverse risk disease, respectively. 2 patients received more than one cycle of chemotherapy (range, 1-2), all developed progressive disease and were removed from study. There were no DLTs at DL 1 or 2. 13 patients enrolled to DL 3 and 3A, 8 had relapsed disease (3 with CR1< 1 year) and 5 were primary refractory. The median age was 53 (range, 19-70) and 4 patients were FLT3-ITD positive. According to ELN genetic risk, 2, 7, 1 and 3 patients had favorable, intermediate I, intermediate II, and adverse risk, respectively. Of the first 3 patients treated at DL 3, one developed grade 3 PN which was a DLT. 3 additional patients were enrolled and 1 patient with a history of anthracycline exposure had a grade 3 decrease in EF. An additional 3 patients were treated, 1 developed grade 3 diarrhea and 1 developed grade 3 PN, both DLT. Given that 8/9 (88%) patients treated at DL 3 achieved a CR or CRi, we chose to further explore the regimen, however, given the concerns for PN, the dose of bortezomib only was modified (DL 3A). 4 additional patients were treated at DL 3A, 2 achieved CR, 1 achieved CRi. No patients have developed PN and there were no DLTs at this dose level. Death within 8 weeks occurred in 8 patients (34.7 %) due to sepsis (1), pneumonia (1), enterococcus bacteremia (1), or progressive disease (5). Overall the objective response rate was 47.8% (11/23), however the CR/CRi response in those who received bortezomib, midostaurin and chemotherapy was 84.6% (11/13). The CR/CRi rate in those with a FLT3 mutation was 75% (3/4). 7 patients have gone on to receive an allogeneic SCT. ConclusionsThe combination of bortezomib, midostaurin and intensive chemotherapy was active in this group of patients with relapsed/refractory AML. DLT of PN was observed, however with a decrease in dose and frequency of bortezomib this has not recurred. Correlative studies to evaluate bortezomib and midostaurin in modulating tyrosine kinase activity are ongoing. Off label useBortezomib in AML Disclosures:Off Label Use: Bortezomib in AML.

Obinutuzumab (GA101), a Novel Type II Glycoengineered CD20 Antibody, Significantly Induced Programmed Cell Death In Primary Mediastinal B-Cell Lymphoma (PMBL): Obinutuzumab May Be a Future Potential Targeted Agent For The Treatment Of PMBL

BackgroundPrimary Mediastinal large B-cell lymphoma (PMBL) is a rare form of Non Hodgkin Lymphoma (NHL) representing 2% of mature B-cell non-Hodgkin lymphoma in patients less than 18 years of age (Lones/Cairo et al., JCO, 2000; Burkhardt et al., BJH, 2005). PMBL has similar histo-pathological features along the biologic spectrum between Diffuse Large B-Cell Lymphoma (DLBCL) and classical HL (cHL) (Abramson et al., Blood, 2005). Gene expression studies suggested that the molecular signature of PMBL had a striking resemblance to the expression profile of cHL (Rosenwald et al., J Exp Med, 2003). We have recently reported that a significant decrease in EFS among children and adolescent PMBL patients compared with other stage III non-PMBL pediatric DLBCL patients following FAB/LMB 96 therapy, suggesting that children and adolescent PMBL may be an inherently different B-NHL (Gerrard/Cairo et al., Blood, 2013). Since over 95% of PMBL express CD20, targeting the CD20 receptor with a CD20 antibody is of high clinical interest. Obinutuzumab (GA101) is novel glycoengineered anti-CD20 targeted monoclonal antibody recognizing a unique CD20 type II epitope and it has been demonstrated to have greater efficacy in reducing tumor size, inducing remission and improving survival in other B-NHL xenograft models (Mössner et al., Blood, 2010). ObjectivesWe hypothesize that obinutuzumab may be a future potential targeted agent for the treatment of PMBL, and therefore, we investigated whether obinutuzumab significantly induces programmed cell death and reduces cell proliferation in PMBL. MethodsCD20+ PMBL, Karpas-1106P cells were obtained from the DSMZ and obinutuzumab was provided by C. Klein, Roche Glycart AG. Total RNA was prepared using Trizol reagent (Invitrogen) from Karpas-1106P cells and cDNA was synthesized using cDNA Synthesis Kit (Quantas). qRT-PCR was performed using CFX96 Real-time system with SsoFast Supermix (Bio-rad). Relative quantification (ddCt) of mRNA expression of CD20 genes was determined by normalizing to GAPDH. Antibodies for western blotting were obtained from Cell signaling Tech. and Santa Cruz Bio., respectively. MTS (Promega) and Caspase 3/7 assay (Promega) were employed for cell proliferation and apoptosis assay with treatment of obinutuzumab (1, 10, 20 and 100ug/ml) at every 24hours. Cell death was assayed by FACS (MoFlo XDP Sorter, Beckman Coulter) of FITC-Annexin V and propidium iodide (PI)–stained cells (BD Biosciences) according to the manufacturer's instruction. Statistical significance was determined by one-tailed Student t-test. ResultsWe observed a significantly higher expression of CD20 mRNA and protein in Karpas-1106P (>5.5-fold, p<0.01 and >2.5-fold, p<0.01) compared to the positive control, CD20+ Raji cells. There was a significant decrease of cell proliferation in obinutuzumab treated Karpas-1106P vs control cells at 24, 48 and 72 hours regardless of the dose of obinutuzumab with 23% reduction (p<0.05), 22% reduction (p<0.05), 24% reduction (p<0.05) and 24% reduction (p<0.05) with 1, 10, 20 and 100ug/ml of obinutuzumab treatment, respectively (Figure 1). A significant increase of Caspase 3/7 activity was observed at day 2 with >3.6-fold (p<0.03), >3.9-fold (p<0.03), >4.0-fold (p<0.02) and >4.2-fold increases (p<0.03) at escalating doses of obinutuzumab treatment, respectively compared to control (Figure 2). Treatment of PMBL cells with obinutuzumab (10 ug/ml) for 48 hours, CD20 expression were significantly decreased 26% (p<0.05) and 10% (p<0.05) in mRNA expression and protein levels, respectively. There was a significant increase of cell death in obinutuzumab treated Karpas-1106P compared to control (PBS) (37.8 vs 1.2%, p<0.02) using Annexin V-binding assay. Additionally, we assessed the expression of the pro-apoptotic gene, Bcl-xL and anti-apoptotic gene, Bax at both the mRNA and protein level. There were significant down-regulation of Bcl-xL (10% reduction, p<0.04) at both of the mRNA and protein levels, and a significant decrease in mRNA expression of Bax (30% reduction, p<0.02). [Display omitted] [Display omitted] ConclusionsObinutuzumab induced a significant increase in PMBL cell death and may be a future potential targeted agent for the treatment of PMBL lymphomas. Future studies will focus on the in vivo effects of obinutuzumab in a NOD/SCID PMBL xenograft mouse model. Disclosures:No relevant conflicts of interest to declare.

Abstract 569: Both Young and Old ACE2-transgenic Mice are More Resistant to Cardiac Dysfunction Induced by Myocardial Infarction

Background: Increased activity of the classic renin-angiotensin system (RAS) is associated with cardiovascular diseases (CVD) such as heart failure and hypertension. Angiotensin-converting enzyme 2 (ACE2) is reported to provide a protective role in CVD. In addition, there is a shift in the balance of the ACE2/Angiotensin-(1-7) axis towards the ACE/Angiotensin II axis during aging, making the cardiovascular system more vulnerable to damages. We hypothesized that global ACE2-overexpression could correct the imbalance between the two axes by providing a cardiovascular protective role against heart failure both in young and aged mice. Methods: Both young (10 weeks old) and old (12 months old) ACE2-transgenic and wild type (WT) mice underwent myocardial infarction surgery. Cardiac function was measured using echocardiography four weeks after MI. The number of circulating inflammatory cells (CD11b) in the blood for these animals was also measured using flow cytometry. Results: In the MI animals, the WT-old mice had a significant reduction in ejection fraction (47.67±4.23%: from 72.63±8.52% to 24.96±4.29%). However, the reduction in EF for ACE2-old mice was 37% lower (26.26±1.90%: from 60.06±7.56% to 29.18±5.66%) than that of WT-old mice. Meanwhile, the MI-induced decrease in EF of 14.60±1.56% for ACE2-young mice (from 59.09±8.38% to 44.49±6.82%) also was significantly less than the 26.97±1.21% reduction observed in WT-young mice (from 53.23±7.67% to 26.26±6.46%). Thus, ACE2 overexpression provides some protection of cardiac function from ischemia-induced injury in both young and old animals. Moreover, the circulating monocytes and macrophages in the blood of ACE2-young MI mice (22.65±6.86%) was also less than which observed in the WT control (31.37±4.90%), suggesting that there is less cardiac damage in these animals. Conclusions: Collectively, our observation suggests that global ACE2 overexpression has a consistent cardiac protection both in the young and old and as a result, the heart may be more resistant to heart failure damage during aging. This protective effect may be partly due to its systematic anti-inflammatory effect.

Relationship between metabolic and structural remodeling during DCM progression in human

Introduction: Whether switch of energy substrates in dilated cardiomyopathy (DCM) is adaptive or causally related to myocardial pathology is still controversial. Thus the aim of our study was a link between structural and metabolic remodeling during DCM progression to heart failure (HF). Population study and methods. Archival RV tissue samples forty-four patients analysed histopathologically and ultrastructurally were subdivided into three groups according to EF determined at the time of admission: group (I) EF 45-55% (n=14, age 23,23±11,75 years), (II) EF 30-44% (n=15, age 24,67±10,36 years), (III) EF <30% (n=15, age 48,66±13,8 years). RNA from ten frozen samples belonging to mentioned groups was isolated and expression levels of PPARα, FAT/CD36, GLUT-4, and CPT-1 investigated. Results: Analysis revealed that with decrease of EF from 45-55% to about 35% was related to increased tissue fibrosis (from 6,15±5,45% to 8,29±5,26%) and cardiomyocytes hypertrophy (from diameter of 17,55±8,07 μm to 18,24±4,62 μm) with accompanying slight reduction of contractile apparatus and increase of mitochondria number and glycogen abundance, and transition of desmin expression from normal pattern to aggregates are characteristic features. Parallel, increase of PPARα and CPT-1, and GLUT-4 and decrease of FAT/CD36 gene expression was observed. While, in myocardial tissues of hearts with decreased EF to <30% was observed significant increase of fibrosis (14,23±12,4%), and not much progress in cardiomyocytes hypertrophy (cells diameter 18,75±4, 44 μm) but significant cell remodeling (related to significant loss of myofibrils, and glycogen, and mitochondria, and desmin cytoskeleton expression, and lipid droplets occurrence). These was accompanying by increased expression of genes - PPARα and FAT/CD36 and CPT-1 comparing to group II about 50% and 150% and 20% respectively, and lowered expression of GLUT4 gene. Conclusions: Obtained data reveled that switch of energy substrates precede cardiomyocytes structural disturbances in DCM. Furthermore, in early disease state glucose metabolism is activated probably to compensate decreased fatty acid transport, particularly that reserve of intracellular glucose is available. While, increase of PPARα and FAT/CD36 genes in later disease stage together with decrease of mitochondria number may lead to toxic lipids storage and cell injury. These data suggest the energetic metabolism as therapeutic purpose to prevent cardiac remodelling in DCM.

α-Lipoic acid attenuates LPS-induced cardiac dysfunction through a PI3K/Akt-dependent mechanism

Myocardial dysfunction is an important manifestation of sepsis/septic shock. Activation of Phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt) signaling pathway has been shown to improve cardiac performance during sepsis/septic shock. We have reported previously that α-lipoic acid (LA) activates PI3K/Akt pathway in neuronal cells. It is possible, therefore, that treatment with LA will attenuate cardiac dysfunction during sepsis/septic shock through a PI3K/Akt-dependent mechanism. To test this possibility, we treated mice with LA prior to lipopolysaccharide (LPS) challenge. Cardiac function was analyzed by echocardiography 6h after LPS challenge. LPS significantly suppressed cardiac function as evidenced by decreases in EF% and FS% in mice. However, LA pretreatment significantly attenuated cardiac dysfunction following LPS challenge. LA pretreatment also improved survival in LPS-challenged mice. Furthermore, LA markedly attenuated the LPS-induced inflammatory response in myocardium, as evidenced by decreases in the upregulation of VCAM-1, ICAM-1 and iNOS, as well as myocardial leucocytes infiltration. Moreover, LPS challenge significantly decreased the phosphorylation levels of Akt and Gsk-3β, which was prevented by LA pretreatment. More importantly, inhibition of PI3K/Akt signaling by Wortmannin (WM) completely abrogated the LA-induced protection in cardiac dysfunction following LPS challenge. Collectively, our results demonstrated that LA improved cardiac function during endotoxemia. The mechanism was through, at least in part, preserved activation of the PI3K/Akt signaling.

Overexpression of CuZnSOD in median preoptic nucleus improves cardiac function after myocardial infarction in the rat

It has been shown that central overexpression of the superoxide scavenging enzyme, Cu/Zn superoxide dismutase (SOD), in the subfornical organ improves cardiac function in a mouse model of heart failure (HF). A downstream hypothalamic site, the median preoptic nucleus (MnPO), may act as a relay center for reactive oxygen species to act as mediators in the pathophysiology of HF. To test the hypothesis that elevated superoxide in the MnPO is related to the pathophysiology of HF and decreased cardiac function, we injected adenovirus encoding CuZnSOD (AdSOD, n=2) or control empty vector (AdE, n=3) into the MnPO of normal rats. Subsequently, rats were subjected to coronary artery ligation to create a myocardial infarct (MI) of the left ventricle. Cardiac function was monitored at 2 week intervals via echocardiography. Upon completion, rat brains were examined for SOD expression in MnPO via immunofluorescence and histopathological analyses of cardiac infarct size were conducted. Baseline (EF) ejection fractions (%) of AdSOD and AdE rats were 62±4 and 67±2, respectively. Two weeks after MI, EF was significantly decreased in both groups of rats (AdSOD: 38±3, AdE: 48±4). In contrast by 6 weeks post MI, EF had improved to 52±1 in AdSOD rats yet was only 41±6 in AdE rats. In conclusion, despite decreases in EF early after MI, overexpression of SOD in the MnPO was related to an improvement in left ventricular function 6 weeks post MI.

Echocardiographic Predictors of Early Postsurgical Myocardial Dysfunction in Pediatric Patients With Aortic Valve Insufficiency

In chronic aortic insufficiency (AI), left-ventricular (LV) dysfunction must be detected early to allow timely surgery. Strain and strain rate have been used for this purpose in adults, but the value of this method in pediatric AI has not been established. Forty patients with moderate to severe AI were included in this retrospective study. LV function was assessed by strain analysis and conventional echocardiography both before and after surgery. Of the 32 patients with preserved preoperative ejection fraction (EF; >50 %), 8 had postoperative dysfunction (<50 %). Mean conventional indices of global LV systolic performance for the entire cohort of patients with AI were predominantly in the normal range before surgery. Preoperative values for LV global longitudinal strain (GLS) and strain rate (GLSr) were normal. After surgery, there was a significant decrease in shortening and EF. There was a significant decrease from preoperative to postoperative values for both GLS (-16.07 ± 3.82 vs. -11.06 ± 3.88; p < 0.0001) and GLSr (-0.89 ± 0.24 vs. -0.72 ± 0.27; p = 0.0021). A preoperative GLS of -15.3 (AUC = 0.83, CI = 0.69-0.98, p < 0.0001) and a GLSr of -0.79/s (AUC = 0.86, CI = 0.73-0.98, p < 0.0001) were determined to be predictors of early postoperative dysfunction after surgical repair of moderate to severe AI. A preoperative GLS value of ≤-15.3 and GLSr value of -0.79/s or less are predictors of postoperative ventricular dysfunction, which is defined by EF <50 %. GLS and GLSr value determination may be useful as part of the echocardiographic assessment AI and may help determine the optimal timing of surgery in pediatric patient with at least moderate AI.

Evaluation of the Effect of Photoselective Vaporization of the Prostate on Sexual Function in a Prospective Study: A Single Center Experience of 150 Patients.

Abstract Background and Purpose: Photoselective vaporization of the prostate (PVP) has emerged as a minimally invasive alternative to transurethral resection of the prostate for treatment of benign prostate hyperplasia (BPH) with lower urinary tract symptoms (LUTS). Very few prospective studies have addressed the effect of PVP on sexual function. We performed this prospective study to assess the short-term effect of PVP on erectile function in patients who present with LUTS secondary to BPH. Patients and Methods: There were 150 consecutive patients who presented with LUTS secondary to BPH who underwent PVP and who were enrolled prospectively in this study. Patients were categorized in two groups: Group A, International Index of Erectile Function (IIEF) ≥19; group B, IIEF <19. PVP was performed by using an 80 W GreenLight laser. Patients preoperative, perioperative, and follow-up data were recorded. Erectile function was assessed preoperatively and postoperatively at 1, 3, 6, and 12 months using IIEF-5. In follow-up, Ultrasonography of the kidneys, ureters, and bladder for residual prostate and postvoid residual urine, uroflowmetry, kidney function tests, and urine culture were performed at each visit. Cystoscopy was performed at 3 months. Data were analyzed statistically. Results: The two patient groups were comparable in their preoperative and perioperative data. Both groups showed improvement in International Prostate Symptom Score, maximum flow rate, quality of life, and decrease in postvoid residual urine at 1, 3, 6, and 12 months with no statistical significance. IIEF-5 scores in Group A, however, were 21.1, 19.2, 17.1, 16.2, and 16.1, respectively, at preoperative, postoperative month 1, 3, 6, and 12, respectively (P=0.02). IIEF-5 scores in Group B were 13.2, 12.1, 11.3, 11.2, and 10.9, respectively, at preoperative, postoperative month 1, 3, 6, and 12, respectively (P=0.45). Conclusions: In patients with normal preoperative erectile function, PVP resulted in significant decrease in EF up to follow-up of 1 year. In patients with preoperative ED, however, PVP did not significantly decrease EF.

Effect of grading characteristics on the undrained shear strength of sand: review with new evidences

In this paper, the shear strength of saturated pure sand and sand-silt mixture is evaluated by monotonic un- drained triaxial tests that were carried out on reconstituted specimens at same relative densities and a constant confin- ing pressure (σ30300 kPa). The test results were used to conclude on the effect of low non-plastic contents (0-20 %) and grading characteristics on the liquefaction resistance of the sand. The test results indicate that the undrained residual strength reduced with the increase of non-plastic fine con- tent. Also, shear strength of gap-graded sand mixed with low non-plastic fine content increases with decrease in ef- fective size (D50). In other words, in this state, we can use the D50 as a parameter to control of silty sand's undrained resistance. Besides, the undrained residual strength of pure sand specimens with same effective size increases due to increase of coefficient of uniformity (Cu).

Abstract 18: Synoviolin, an E3 Ubiquitin Ligase, Modulates Cardiac Myocyte Size and Restores Heart Function in Hypertrophic Cardiomyopathy

The endoplasmic reticulum (ER) is essential for protein homeostasis, or proteostasis, which governs the balance of the proteome. In addition to secreted and membrane proteins, proteins bound for many other cellular locations are also made on ER-bound ribosomes, emphasizing the importance of protein quality and quantity control in the ER. Unlike cytosolic E3 ubiquitin ligases studied in the heart, synoviolin/Hrd1, which has not been studied in the heart, is an ER transmembrane E3 ubiquitin ligase, which we found to be upregulated upon protein misfolding in cardiac myocytes. Given the strategic location of synoviolin in the ER membrane, we addressed the hypothesis that synoviolin is critical for regulating the balance of the proteome, and accordingly, myocyte size. We showed that in vitro, adenovirus-mediated overexpression of synoviolin decreased cardiac myocyte size and protein synthesis, but unlike atrophy-related ubiquitin ligases, synoviolin did not increase global protein degradation. Furthermore, targeted gene therapy using adeno-associated virus 9 (AAV9) showed that overexpression of synoviolin in the left ventricle attenuated maladaptive cardiac hypertrophy and preserved cardiac function in mice subjected to trans-aortic constriction (AAV9-control TAC = 22.5 ± 6.2% decrease in EF vs. AAV9-synoviolin TAC at 6 weeks post TAC; P&lt;0.001), and decreased mTOR activity. Since calcium is a major regulator of cardiac myocyte size, we examined the effects of synoviolin gain- or loss-of-function, using AAV9-synoviolin, or an miRNA designed to knock down synoviolin, respectively. While synoviolin gain-of-function did not affect calcium handling in isolated adult myocytes, synoviolin loss-of-function increased calcium transient amplitude (P&lt;0.01), prolonged spark duration (P&lt;0.001), and increased spark width (P&lt;0.001). Spark frequency and amplitude were unaltered upon synoviolin gain- or loss-of-function. Whereas SR calcium load was unaltered by synoviolin loss-of-function, SERCA-mediated calcium removal was reduced (P&lt;0.05). In conclusion, our studies suggest that in the heart, synoviolin is 1) a critical component of proteostasis, 2) a novel determinant of cardiac myocyte size, and 3) necessary for proper calcium handling.

A multicenter, open-label phase II trial of liposomal doxorubicin (LD) and docetaxel (DX), followed by concurrent chemotherapy and radiation therapy in locally advanced high-risk, early breast cancer (HRBC).

e11041 Background: Taxanes and anthracyclines (ANs) are among the most active agents in the treatment of breast cancer. However, ANs are associated with dose-related cardiac toxicity, which can lead to progressive myocardial damage. Non-pegylated liposome-encapsulated doxorubicin (Myocet®), has been shown to significantly reduce cardiac toxicity even though providing comparable antitumor efficacy. Primary endpoint of this phase II study was the assessment of early and late cardiac toxicity of young patients with HRBC treated with LD and DX. Secondary end-points were the incidence of hematological toxicity, time to progression and overall survival in this cohort of patients. Methods: From 05-2006 to 10-2009, 60 HRBC patients were entered into the study. They were treated with LD 60 mg/m2 and DX 75 mg/m2 every three weeks for six courses. Hematological growth factors were given when indicated. Cyclophosphamide 600 mg/m2, methotrexate 60 mg/m2 and 5-fluorouracil 600 mg/m2 were given for six courses concomitantly with radiation therapy to the breast, axilla and supraclavicular nodes. After chemo-radiation therapy, a hormonal therapy was given to estrogen receptors positive (ER+) patients and trastuzumab to Herb-2 positive patients. LVEF was evaluated at baseline and after 6 months, thereafter, up to 5 years. Results: Median age was 49 years. Mean number of positive axillary nodes was 4.9 (range 0-20). 70% were ER+, 30% were ER - and progesterone receptor (PGR) negative. Median Ki-67 was 30% (range 20%-100%), 18% of patients were Herb-2 positive and 18% of patients had pre-existing hypertension. Delivered dose was 95.7% of the planned dose. No patient showed neither a decrease of EF &gt; 10%, nor congestive heart failure. Febrile neutropenia was observed in 12% of patients. At 44 months of median follow-up, 86% of patients were disease-free and 94% were alive. Conclusions: Chemotherapy with LD and DX has shown low cardiac toxicity and high activity in this group of HRBC patients.