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A number of intracellular structural/nonstructural proteins and receptors expressed by cardiac myocytes have been identified as putative targets of autoimmune response in subsets of patients classified as those suffering from idiopathic dilated cardiomyopathy (IDCM). At present it is difficult to determine whether such antibodies are a primary or secondary phenomenon; however, it is becoming increasingly clear that the presence of such autoantibodies against cardiac tissues most likely does contribute to the pathogenic process. This view is supported by the results of immunoglobulin apheresis of dilated cardiomyopathy (DCM) patients, which appears to lead to significant improvement of cardiac function at least in a select group of such patients. The concept of “molecular mimicry” between proteins of infectious agents and cardiac tissue proteins and receptors appears to provide the most convincing evidence for an infectious etiology for at least a subgroup of DCM patients. Among the autoantigens, the alpha chain of cardiac myosin and the beta-1-adrenoreceptor seem to provide the most consistent data as targets of autoimmune responses. The data taken together clearly suggest that as previously suspected, human DCM is a complex heterogeneous disease and is likely a result of host genetics, cardiac tissue insult in the form of an infectious process or a cardiotoxic agent, a dysregulation of immune regulatory mechanisms, and metabolic/contractile abnormalities. How each of these interplays in different DCM patients at different stages of the disease process influences the complexity of the results one obtains in studying this heterogeneous disease. Murine models of coxsackie virus infection and experimental immunization of mice with cardiac myosin in inbred and various gene knockout strains of mice have provided important clues. Similarly, the fact that immunization of rodents with synthetic myosin peptides that resemble the peptides present on the outer membrane protein of Chlamidya and induce cardiac dysfunction adds to such models. Finally, the observation that mice that have targeted gene deletions for PD-1 within T cells and targeted gene deletions for the intracellular signaling molecule STAT-3 to cardiac tissue, both of which develop heart failure, provides yet other pieces to the puzzle of human-dilated cardiomyopathies. Attempts to identify which of these models serve as surrogates for which subset of DCM patient remain a future challenge. Identification of antibodies against cardiac myosin and the beta-1-adrenoreceptor protein may provide an initial means to segregate patients into those that present an autoimmune component and those that do not in efforts to identify groups of patients that could benefit best from therapies aimed at treating these patients for the autoimmune process.

Background— The in vivo regeneration capacity of decellularized heart valve grafts is still controversial. The aim of this study was to evaluate function, morphological changes, and cellular composition of decellularized versus re-endothelialized ovine pulmonary valves (PV) after implantation into lambs for 1 or 3 months. Methods and Results— PV (n=21) were decellularized using detergents. Twelve PV were repopulated with autologous jugular veins endothelial cells (ECs) in a dynamic pulsatile bioreactor under simulated physiological conditions. Morphological evaluation before implantation included histological stainings (H&E, Movat-pentachrome, von-Kossa, DAPI), immunostainings (anti-perlecan, anti-eNOS, anti-procollagen-I, anti-SM-α-actin), electron microscopy (EM), and DNA extraction. Decellularization led to cell-free scaffolds with preserved extracellular matrix (ECM) including basement membrane. Reseeded PV (n=5) were completely covered with ECs expressing endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). The function of orthotopically implanted decellularized and re-endothelialized PV (n=7, each) was analyzed after 1 and 3 months by echocardiography and revealed no differences in competence between both groups. A confluent EC monolayer expressing eNOS/vWF was only found in re-endothelialized PV but not in decellularized PV, whereas the valve matrices were comparable repopulated with interstitial cells expressing SM-α-actin and procollagen-I. More thrombotic and neointima formations were observed in decellularized PV. No signs of calcification were detected in both PV types. Conclusion— In vitro re-endothelialization of detergent-decellularized valves with autologous ECs under simulated physiological conditions significantly improves total EC valve coverage 3 months after implantation, whereas the valve repopulation with interstitial cells in vivo occurs most likely by cell migration inside the scaffold.

The intracellular adaptor protein SH3P7 is the mammalian ortholog of yeast actin-binding protein 1 and thus alternatively named as mAbp1 (or HIP55). Structural properties, biochemical analysis of its interaction partners and siRNA studies implicated mAbp1 as an accessory protein in clathrin-mediated endocytosis (CME). Here, we describe the generation and characterization of mice deficient for SH3P7/mAbp1 owing to targeted gene disruption in embryonic stem cells. Mutant animals are viable and fertile without obvious deficits during the first weeks of life. Abnormal structure and function of organs including the spleen, heart, and lung is observed at about 3 months of age in both heterozygous and homozygous mouse mutants. A moderate reduction of both receptor-mediated and synaptic endocytosis is observed in embryonic fibroblasts and in synapses of hippocampal neurons, respectively. Recycling of synaptic vesicles in hippocampal boutons is severely impaired and delayed four-fold. The presynaptic defect of SH3P7/mAbp1 mouse mutants is associated with their constricted physical capabilities and disturbed neuromotoric behaviour. Our data reveal a nonredundant role of SH3P7/mAbp1 in CME and places its function downstream of vesicle fission.

Transthoracic echocardiography is the predominant diagnostic tool to evaluate systolic and diastolic cardiac function noninvasively in mice. It is known that systolic function is substantially influenced by anesthetic agents used for sedation during echocardiography. However, the effect on diastolic function has not been investigated yet. The following study was conducted to evaluate the influence of different agents on diastolic left ventricular function in mice.The effect of ketamine/xylazine (K/X), ketamine/midazolam (K/M), and tribromoethanol (TBE, Avertin) on diastolic function was measured 5, 15, and 25 minutes after the onset of anesthesia. Ratio of peak early-to-late myocardial diastolic velocities (Ea/Aa; determined by tissue Doppler imaging; TDI), ratio of peak transmitral early (E)- and late-diastolic velocity (E/A), deceleration time (DT), and isovolumic relaxation time (IVRT) correlated significantly with heart rate (HR). Overall, increasing HR contributed to a decrease of E/A-, Ea/Aa ratio, IVRT, and DT, whereas agents characterized by the strongest variation of HR (K/M and TBE) were associated with the greatest effect on diastolic function.Left ventricular diastolic function in mice, determined by echocardiography, is dependent on anesthetic agent and timing of measurements after onset of anesthesia.

BACKGROUND Transplantation of bone marrow derived adult stem cells (BMC) improves cardiac function after acute myocardial infarction (MI). However, the cell population mediating myocardial recovery and the fate of the transplanted cells are still controversial. AIMS We determined the effects of Sca-1+ c-kit+ lin- haematopoietic BMC on cardiac function after MI and the cell fate after transplantation. METHODS Sca-1+ c-kit+ lin- BMC of male donor C57BL/6 mice were transplanted by intravenous injection into syngenic females after permanent MI. LV dimensions and function were determined by echocardiography and cardiac magnetic resonance imaging, transplanted BMC were identified by Y chromosome DNA in situ hybridization. RESULTS BMC treatment completely prevented LV dilation (LV end-diastolic volume BMC 70 +/- 16 microl vs. control 122 +/- 41 microl; p < 0.05) and improved fractional shortening (BMC 22.9 +/- 8% vs. control 15.4 +/- 8.4%; p < 0.05) and ejection fraction BMC 68.2 +/- 6.6% vs. control 52 +/- 14.3%; p < 0.05) as early as 3 days after transplantation, but did not decrease infarct size (BMC 27 +/- 6% vs. control 28 +/- 7%, p = n.s.). After 4 weeks, only sporadic cells of male origin were identified in infarcted hearts (< 0.01% of periinfarct cells). CONCLUSION Intravenous injection of Sca-1+ c-kit+ lin- in BMC after MI improves LV dimensions and function without evidence for long term engraftment.

Aims Peripartum cardiomyopathy (PPCM) is a disorder of unknown aetiology with a course and outcome that is largely unpredictable. We evaluated the prognostic role of multiple inflammatory markers in the plasma of a large cohort of African patients with PPCM. Methods and results The study of 100 patients with newly diagnosed PPCM was single-centred, prospective, and longitudinal. Clinical assessment, echocardiography, and blood analysis were done at baseline and after 6 months of standard therapy. Inflammatory markers were measured at baseline only. Fifteen patients died. Left ventricular ejection fraction (LVEF) improved from 26.2±8.2 to 42.9±13.6% at 6 months ( P 50%) was only observed in 23%. Baseline levels of C-reactive protein correlated positively with baseline LV end-diastolic (rs=0.33, P =0.0026) and end-systolic (rs=0.35, P =0.0012) diameters and inversely with LVEF (rs=−0.27, P =0.015). Patients who died presented with significantly lower mean EF and higher Fas/Apo-1 plasma values ( P

Background— The Jun family of activator protein 1 (AP-1) transcription factors (c-Jun, JunB, and JunD) is involved in fundamental biological processes such as proliferation, apoptosis, tumor angiogenesis, and hypertrophy. The role of individual AP-1 transcription factors in the stressed heart is not clear. In the present study we analyzed the role of JunD in survival, hypertrophy, and angiogenesis in the pressure-overloaded mouse heart after thoracic aortic constriction. Methods and Results— Mice lacking JunD (knockout [KO]) showed increased mortality and enhanced cardiomyocyte apoptosis and fibrosis associated with increased levels of hypoxia-induced factor-1α, vascular endothelial growth factor (VEGF), p53, and Bax protein and reduced levels of Bcl-2 protein after 7 days of severe pressure overload compared with wild-type (WT) siblings. Cardiomyocyte hypertrophy in surviving KO mice was enhanced compared with that in WT mice. Chronic moderate pressure overload for 12 weeks caused enhanced left ventricular hypertrophy in KO mice, and survival and interstitial fibrosis were comparable with WT mice. Cardiac function, 12 weeks after operation, was comparable among shams and pressure-overloaded mice of both genotypes. In addition, KO mice exposed to chronic pressure overload showed higher cardiac capillary density associated with increased protein levels of VEGF. Conclusions— Thus, JunD limits cardiomyocyte hypertrophy and protects the pressure-overloaded heart from cardiac apoptosis. These beneficial effects of JunD, however, are associated with antiangiogenic properties.

The transcription factor signal transducer and activator of transcription 3 (STAT3) participates in a wide variety of physiological processes and directs seemingly contradictory responses, such as proliferation and apoptosis. The constitutive activation of STAT3 promotes tumor growth and angiogenesis and is associated with drug resistance in cancer therapy. In contrast, in the heart, the down-regulation of STAT3 has been associated with end-stage heart failure in patients. Moreover, multiple studies showed that the activation of STAT3 promotes cardiomyocyte survival and hypertrophy, as well as cardiac angiogenesis, in response to various pathophysiologic stimuli, strongly suggesting that STAT3 is beneficial for the heart. Conditional knockout (STAT3-KO) mice harboring a cardiomyocyte-restricted deletion of STAT3 showed enhanced susceptibility to cardiac injury caused by myocardial ischemia, systemic inflammation, or drug toxicity. STAT3-KO mice were also more prone to the pathogenesis of age-related heart failure. Thus, STAT3 is involved in multiple mechanisms required for the protection of the heart from injury and heart failure. These observations should be taken into account in designing novel therapeutic strategies for the prevention of cardiac failure.

Adverse left ventricular (LV) remodeling after myocardial infarction (MI) is a major cause for heart failure. Molecular modifiers of the remodeling process remain poorly defined. Patients with heart failure after MI have reduced LV expression levels of muscle LIM protein (MLP), a component of the sarcomeric Z-disk that is involved in the integration of stress signals in cardiomyocytes. By using heterozygous MLP mutant (MLP +/— ) mice, we explored the role of MLP in post-MI remodeling. LV dimensions and function were similar in sham-operated WT and MLP +/— mice. After MI, however, MLP +/— mice displayed more pronounced LV dilatation and systolic dysfunction and decreased survival compared with WT mice, indicating that reduced MLP levels predispose to adverse LV remodeling. LV dilatation in MLP +/— mice was associated with reduced thickening but enhanced elongation of cardiomyocytes. Activation of the stress-responsive, prohypertrophic calcineurin–nuclear factor of activated T-cells (NFAT) signaling pathway was reduced in MLP +/— mice after MI, as shown by a blunted transcriptional activation of NFAT in cardiomyocytes isolated from MLP +/— /NFAT-luciferase reporter gene transgenic mice. Calcineurin was colocalized with MLP at the Z-disk in WT mice but was displaced from the Z-disk in MLP +/— mice, indicating that MLP is essential for calcineurin anchorage to the Z-disk. In vitro assays in cardiomyocytes with down-regulated MLP confirmed that MLP is required for stress-induced calcineurin–NFAT activation. Our study reveals a link between the stress sensor MLP and the calcineurin–NFAT pathway at the sarcomeric Z-disk in cardiomyocytes and indicates that reduced MLP–calcineurin signaling predisposes to adverse remodeling after MI.

Background— Accumulating evidence suggests a critical role for increased reactive oxygen species (ROS) production in left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). Increased expression of xanthine oxidase (XO), a major source of ROS, has recently been demonstrated in experimental and clinical heart failure; however, a potential role for LV remodeling processes remains unclear. We therefore studied the effect of long-term treatment with allopurinol, a potent XO inhibitor, on myocardial ROS production and LV remodeling and dysfunction after MI. Methods and Results— Mice with extensive anterior MI (n=105) were randomized to treatment with either vehicle or allopurinol (20 mg · kg −1 · d −1 by gavage) for 4 weeks starting on day 1 after surgery. Infarct size was similar among the groups. XO expression and activity were markedly increased in the remote myocardium of mice after MI, as determined by electron spin resonance spectroscopy. Myocardial ROS production was increased after MI but markedly reduced after allopurinol treatment. Importantly, allopurinol treatment substantially attenuated LV cavity dilatation and dysfunction after MI, as assessed by echocardiography, and markedly reduced myocardial hypertrophy and interstitial fibrosis. Conclusion— The present study reveals a novel beneficial effect of treatment with allopurinol, ie, a marked attenuation of LV remodeling processes and dysfunction after experimental MI. Allopurinol treatment therefore represents a potential novel strategy to prevent LV remodeling and dysfunction after MI.

Background— CCN1, a potent proangiogenic factor, is induced in the vasculature by tissue injury, angiotensin II (Ang II), and growth factor stimulation. Because these conditions occur in myocardial ischemia and pressure overload, we investigated the regulation of CCN1 in cardiomyocytes in vitro and in the heart in vivo. Methods and Results— Ang II, signaling via the angiotensin type 1 (AT 1 ) receptor, and α 1 -adrenergic stimulation with phenylephrine induced CCN1 expression in ventricular cardiomyocytes isolated from 1- to 3-day-old rats. Cell culture supernatant of Ang II–treated cardiomyocytes induced migration of smooth muscle cells, which was abolished by neutralizing antibody to CCN1. Ang II– and phenylephrine-mediated induction of CCN1 expression in cardiomyocytes was completely abolished by inhibition of MEK/extracellular signal–regulated kinases (ERK) or protein kinase C (PKC). Likewise, mechanical stretch induced CCN1 expression in cardiomyocytes, an effect that was prevented by AT 1 receptor blockade or PKC inhibition. Similarly, pressure overload in vivo upregulated myocardial CCN1 expression levels via AT 1 receptor– and PKC-dependent mechanisms. After myocardial infarction in mice, CCN1 expression was strongly induced in both ischemic and remote left ventricular myocardium. Marked CCN1 protein expression was noted in cardiomyocytes of patients with end-stage ischemic cardiomyopathy but was almost absent in nonfailing human myocardium. Conclusions— Pressure overload, ischemia, and neurohormonal factors, such as Ang II or α 1 -adrenergic stimuli, induce myocardial expression of CCN1, a potent proangiogenic factor, supporting the notion that CCN1 may play an important role in the adaptation of the heart to cardiovascular stress.

Sex-determining cascades are supposed to have evolved in a retrograde manner from bottom to top. Wilkins' 1995 hypothesis finds support from our comparative studies in Drosophila melanogaster and Musca domestica, two dipteran species that separated some 120 million years ago. The sex-determining cascades in these flies differ at the level of the primary sex-determining signal and their targets, Sxl in Drosophila and F in Musca. Here we present evidence that they converge at the level of the terminal regulator, doublesex ( dsx), which conveys the selected sexual fate to the differentiation genes. The dsx homologue in Musca, Md-dsx, encodes male-specific (MdDSX(M)) and female-specific (MdDSX(F)) protein variants which correspond in structure to those in Drosophila. Sex-specific regulation of Md-dsx is controlled by the switch gene F via a splicing mechanism that is similar but in some relevant aspects different from that in Drosophila. MdDSX(F) expression can activate the vitellogenin genes in Drosophila and Musca males, and MdDSX(M) expression in Drosophila females can cause male-like pigmentation of posterior tergites, suggesting that these Musca dsx variants are conserved not only in structure but also in function. Furthermore, downregulation of Md-dsx activity in Musca by injecting dsRNA into embryos leads to intersexual differentiation of the gonads. These results strongly support a role of Md-dsx as the final regulatory gene in the sex-determining hierarchy of the housefly.

The transcription factor signal transducer and activator of transcription 3 (STAT3) participates in a wide variety of physiological processes and directs seemingly contradictory responses such as proliferation and apoptosis. To elucidate its role in the heart, we generated mice harboring a cardiomyocyte-restricted knockout of STAT3 using Cre/loxP-mediated recombination. STAT3-deficient mice developed reduced myocardial capillary density and increased interstitial fibrosis within the first 4 postnatal months, followed by dilated cardiomyopathy with impaired cardiac function and premature death. Conditioned medium from STAT3-deficient cardiomyocytes inhibited endothelial cell proliferation and increased fibroblast proliferation, suggesting the presence of paracrine factors attenuating angiogenesis and promoting fibrosis in vitro. STAT3-deficient mice showed enhanced susceptibility to myocardial ischemia/reperfusion injury and infarction with increased cardiac apoptosis, increased infarct sizes, and reduced cardiac function and survival. Our study establishes a novel role for STAT3 in controlling paracrine circuits in the heart essential for postnatal capillary vasculature maintenance, interstitial matrix deposition balance, and protection from ischemic injury and heart failure.

Circulating levels of interleukin (IL)-6 are elevated after myocardial infarction (MI) and associated with increased morbidity and mortality. Its myocardial expression post-MI suggests a pathophysiological role in this condition. To explore the role of endogenous IL-6, we analyzed MI size, left ventricular (LV) remodeling, and mortality after permanent coronary ligation in IL-6 knockout mice (IL-6-/-) and wild-type controls (WT). Six weeks after MI, IL-6-/- and WT had similar mortality rates, MI sizes, LV remodeling, and LV dysfunction in vivo, determined by catheterization. Infarct size 24 h post-MI, shown by 2,3,5-triphenyltetrazolium chloride (TTC) staining, was similar at 24 h. Treatment with exogenous IL-6 did not alter MI size in WT. Infarction resulted in marked phosphorylation of STAT3, without differences between genotypes. Leukemia inhibitory factor (LIF) protein was increased 48 h post-MI in IL-6-/-, and angiotensin II and AT1 receptor (AT1R) protein were strongly increased in IL-6-/- baseline and post-MI, suggesting compensatory up-regulation. Lack of IL-6 does not affect long-term MI size or LV function, remodeling, and survival. In mice lacking IL-6, other members of the IL-6 family such as LIF and other factors signaling via JAK/STAT such as angiotensin may act in a compensatory manner to activate the JAK/STAT pathway, thereby maintaining STAT3 phosphorylation, which is crucial for the cellular effects of IL-6 cytokines.

Introduction Les pathologies cardiovasculaires liees a la grossesse sont associee a un risque majeur de morbi-mortalite. La prise en charge peri-obstetricale de la pre eclampsie a beneficie d’avancees recentes basees sur une meilleure maitrise de sa physiopathologie : desequilibre de la balance angiogenique mis en evidence par l’augmentation du ratio sFlt-1/PlGF dans le plasma maternel. La cardiomyopathie du peripartum (CMPP) est une forme d’insuffisance aigue grave, souvent pendant l’allaitement, qui peut se transformer en choc cardiogenique et necessiter la mise sous assistance ventriculaire et parfois la transplantation cardiaque. Malgre le pronostic grave de cette pathologie, aucun marqueur plasmatique ou urinaire n’est decrit a ce jour pour aider au diagnostic ou evaluer le pronostic. Il est a ce jour prouve sur des modeles animaux et confirme sur des series de patientes atteintes de CMPP que l’inhibition en post partum immediat de la secretion de prolactine (fraction 24 kD) par la bromocriptine, serait associee a un meilleur pronostic cardiaque. Nous emettons l’hypothese que la CMPP est associee a une alteration de la balance angiogenique et de celle de la relaxine-2, facteur placentatire aux proprietes hemodynamiques benefiques chez la femme enceinte. Materiel et methodes Les facteurs Anti (sFlt-1), pro (PlGF or VEGF) angiogeniques et la relaxine-2 ont ete mesures au niveau plasmatiques et tissulaires myocardiques chez des patientes (Afrique du Sud, France, Allemagne) ayant une CMPP. Les malades ont signe un accord ecrit. Resultats Les ratios sFlt-1/PlGF [1,3 (0,9 a 2,8)] et sFlt-1/VEGF [0,6 (0,2 a 1,2)] sont effondres chez les CMPP versus 52,8 (20,6 a 116,2) chez les femmes allaitantes ayant une fonction cardiaque normale. De plus les seuils plasmatiques de sFlt-1/PlGF Discussion Cette etude montre une alteration plasmatique et tissulaire myocardique de la balance angiogenique ainsi que de la secretion de la Relaxine-2, chez les patientes qui developpent une CMPP. L’association d’un bas ratio sFlt-1/PlGF et d’un taux bas de Relaxine-2 plasmatiques sont associes au diagnostic de CMPP. Ces resultats permettront de detecter dans un contexte de peri-partum les patientes atteintes de CMPP, pour instaurer rapidement un traitement par la bromocriptine. Des etudes complementaires pour une evaluation de l’effet de la serelaxine (Relaxine-2 recombinante humaine) seraient a prevoir au cours de la CMPP.

Purpose: Implementation of PTA as standard treatment for patients with myocardial infarction (MI) reduced the mortality in the early phase. However, morbidity due to adverse cardiac remodelling has increased. In MI-patients, serum concentrations of free sialic acids are significantly elevated. Those are released from glycoconjugates by neuraminidases (Neu). Neu play important roles in various biological processes, i.e. inflammation, differentiation and metabolism. Here, we investigate the role and regulation of Neu1 in monocytes/macrophages (Mo/Mϕ) after cardiac ischemia/reperfusion (I/R). Methods & results: Wild type mice undergoing cardiac I/R operation (50 min occlusion of the LAD followed by reperfusion) showed a significant up-regulation of Neu1 mRNA and protein expression in the ischemic area 3 d after I/R compared to sham operated mice (191±40%, p

Epigenetic programming within the cardiac stem cell (CSC) niche controls CSC specification and differentiation, but the identity of putative mediators is poorly characterized. To dissect these mechanisms, we used primary and clonally expanded Sca-1+/c-kit+ CSC from murine adult hearts and stimulated cardiomyocyte differentiation upon culture in a differentiation medium (DIFF) containing 5'-Azacytidine and transforming growth factor β1 or co-cultured with rat cardiomyocytes. With this model, we previously showed that inducible deletion of β-catenin enhanced CSC differentiation in vitro and in vivo. Accordingly, we detected a constitutive activity of Wnt/b-catenin pathway in undifferentiated CSC and a downregulation of Wnt target genes (in % of ctl: Axin2: 36±4%; Snai2: 46±7%; n=3; p

Purpose: Patients suffering from heart failure or advanced cancer share clinical symptoms including limited exercise capacity, shortness of breath and early fatigue, and develop skeletal and cardiac muscle atrophy. The systemic effects of cancer on the heart are poorly understood. We investigated molecular and metabolic changes of the heart in a B16F10 melanoma mouse cancer model. Methods: B16F10 melanoma tumor was induced by intraperitoneal implantation of B16F10 cells. Morphological, histological and molecular biological (Western Blot, qRT-PCR) analyses, echocardiography and glucose uptake by FDG-PET were carried out 3 weeks later. Results: Tumor growth was confirmed morphologically and by FDG-PET analysis. Tumor-bearing wildtype (C57BL6) mice displayed cardiac atrophy and high mortality (control: 0% vs tumor mice: 66%, P

Despite advances in both targeted therapies with ABL-specific tyrosine kinase inhibitors and in allogeneic stem cell transplantation, BCR-ABL positive acute lymphoblastic leukemia (ALL) remains a very high-risk disease, necessitating the development of novel treatment strategies. miRNAs are small non-coding RNAs which regulate gene expression posttranscriptionally in a sequence-specific manner. miRNAs usually repress the expression of many target genes. We hypothesized that miRNAs may help to identify potential therapeutic targets if (i) they are expressed in a disease-specific manner and if (ii) modulating their expression induces a desired phenotype, such as apoptosis of tumour cells, in appropriate experimental models. Based on our observation that miR∼17-92-encoded miRNAs are significantly less abundant in primary BCR-ABL-positive as compared to negative ALL-cells, we studied the expression and function of miRNAs encoded by the miR∼17-92 derivative miR∼17-19b in a murine pro-B-cell line with inducible BCR-ABL-expression (TonB). Induction of BCR-ABL expression in TonB cells reduced endogenous miR-17, miR-18a, and miR-19 by 2 to 3.5-fold, confirming that expression of the miR∼17-92 cluster is controlled by BCR-ABL. Interestingly, over-expression of miR∼17-19b by lentiviral gene transfer led to a substantial induction of apoptosis in TonB cells in a BCR-ABL-dependent manner. To identify potential miRNA targets, we used a proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) followed by liquid chromatography and mass spectroscopy (LC-MS) in miR∼17-19b transgenic TonB cells. Several apoptosis-related proteins were differentially expressed including Bcl2, an established inhibitor of mitochondrial pro-apoptotic pathways. The miRNA target prediction program RNA22 predicted several miR∼17-19b miRNA-binding sites within both murine and human Bcl2 mRNA, and we demonstrated direct miRNA binding to Bcl2 mRNA by luciferase reporter and anti-AGO2 RIP chip analyses. As with miR∼17-19b over-expression, Bcl2 specific RNAi strongly induced apoptosis in murine TonB and the human BCR-ABL-positive cell lines BV-173, Tom1 and SupB15. BCR-ABL positive human ALL-cell lines were also more sensitive than negative ones to pharmacological BCL2 inhibition with the BH3 mimetic ABT 737. In addition, inhibition of BCL2 by ABT 737 and BCR-ABL kinase activity by Imatinib exert different anti-leukemic effects with differential impact on miR∼17-92 miRNA-expression. To assess the therapeutic potential of BCL2 inhibition we used a xenotransplantation assay with real time in vivo monitoring of drug therapies by bioluminescent imaging. ABT-737 treatment substantially inhibited expansion of luciferase-expressing human primary BCR-ABL-positive ALL xenografts in NOD/LtSz-scid IL-2Rγ null (NSG) mice and significantly lengthened their median survival. Taken together, our data identify BCL2 as a therapeutic target of particular relevance in BCR-ABL-positive ALL and indicate involvement of miR∼17-92-encoded miRNAs in regulation of apoptosis in these cells. The validity of this miRNA-based approach to identify potential drug targets is demonstrated by the efficacy of the BCL2 inhibitor ABT-737 in an in vivo model of human BCR-ABL positive ALL, suggesting that BCL2 inhibition should be considered for early phase clinical testing as a strategy to improve disease outcomes. Disclosures: No relevant conflicts of interest to declare.

Abstract —Inflammatory processes involve both synthesis of inflammatory cytokines, such as interleukin-6 (IL-6), and the activation of their distinct signaling pathways, eg, the janus kinases (JAKs) and signal transducers and activators of transcription (STAT). Superoxide (O 2 − ) anions activate this signaling cascade, and the vasoconstrictor angiotensin II (Ang II) enhances the formation of O 2 − anions via the NAD(P)H oxidase system in rat aortic smooth muscle cells. Ang II activates the JAK/STAT cascade via its type 1 (AT 1 ) receptor and induces synthesis and release of IL-6. Therefore, we investigated the role of O 2 − anions generated by the NAD(P)H oxidase system on the Ang II activation of the JAK/STAT cascade and its impact on IL-6 synthesis. Ang II stimulation of rat aortic smooth muscle cells induced a rapid increase in O 2 − anions determined by laser fluoroscopy, which can be abolished by DPI, a flavoprotein inhibitor. Ang II–induced phosphorylation of JAK2, STAT1α/β, STAT3, and IL-6-synthesis can be abolished by DPI, as determined by immunoprecipitations and Northern blot analysis. Electroporation of neutralizing antisera targeted against p47 phox , a NAD(P)H oxidase subunit, abolished Ang II–induced JAK/STAT activation and IL-6 synthesis. Inhibition of JAK2 by its inhibitor AG490 (10 μmol/L) blocked not only JAK2 activation but also IL-6 synthesis. These results suggest that stimulation of the JAK/STAT cascade by Ang II requires O 2 − anions generated by the NAD(P)H oxidase system, and O 2 − anion–dependent activation of the JAK/STAT cascade seems to be additionally involved in Ang II–induced IL-6 synthesis. Thus, Ang II–induced inflammatory effects seem to require O 2 − anions generated by the NAD(P)H oxidase system.

Peripartum cardiomyopathy (PPCM) is a rare disease of unclear etiology with a frequent poor outcome, despite optimal medical therapy. Recent experimental data implicate a causal role of prolactin. We report a patient with PPCM who responded well to treatment with Bromocriptine in addition to standard therapy of heart failure.

To the Editor: Postpartum cardiomyopathy (PPCM) is a disease of unknown origin and exposes women to a high risk of mortality after delivery despite optimal medical therapy ([1][1]). Prolactin is up-regulated postpartally where it induces lactation and promotes reshaping of the uterus. Prolactin

Sex-lethal (Sxl) is the master switch gene for somatic sex determination in Drosophila melanogaster. In XX animals, Sxl becomes activated and imposes female development; in X(Y) animals, Sxl remains inactive and male development ensues. A switch gene for sex determination, called F, has also been identified in the housefly, Musca domestica. An active F dictates female development, while male development ensues when F is inactive. To test if the switch functions of Sxl and F are founded on a common molecular basis, we isolated the homologous Sxl gene in the housefly. Though highly conserved in sequence, Musca-Sxl is not sex-specifically regulated: the same transcripts and protein isoforms are expressed in both male and female animals throughout development. Musca-Sxl is apparently not controlled by the primary sex-determining signal and, thus, is unlikely to correspond to the F gene. Ectopic expression of Musca-SXL protein in Drosophila does not exert any noticeable effects on the known target genes of endogenous Sxl. Instead, forced overexpression of the transgene eventually results in lethality of both XY and XX animals and in developmental abnormalities in some escaper XY animals. Similar results were obtained with the Sxl homologue of Ceratitis capitata (Saccone, G., Peluso, I., Artiaco, D., Giodano, E., Bopp, D. and Polito, L. C. (1998) Development 125, 1495–1500) suggesting that, in these non-drosophilid species, Sxl performs a function different from that in sex determination.

Dosage compensation is a regulatory process that insures that males and females have equal amounts of X-chromosome gene products. In Drosophila, this is achieved by a 2-fold enhancement of X-linked gene transcription in males, relative to females. The enhancement of transcription is mediated by the activity of a group of regulatory genes characterized by the male-specific lethality of their loss-of-function alleles. The products of these genes form a complex that is preferentially associated with numerous sites on the X chromosome in somatic cells of males but not of females. Binding of the dosage compensation complex is correlated with a significant increase in the presence of a specific histone isoform, histone 4 acetylated at Lys16, on this chromosome. Experimental results and sequence analysis suggest that an additional gene, males-absent on the first (mof), encodes a putative acetyl transferase that plays a direct role in the specific histone acetylation associated with dosage compensation. The predicted amino acid sequence of MOF exhibits a significant level of similarity to several other proteins, including the human HIV-1 Tat interactive protein Tip60, the human monocytic leukemia zinc finger protein MOZ and the yeast silencing proteins SAS3 and SAS2.

Purpose Mortality in adult patients requiring ECMO support is high. We aimed to identify biomarkers and potential pathophysiological mechanisms triggered by ECMO that could be responsible for adverse outcome. Oxidative stress during ECMO is increased and unbalanced oxidative stress is linked to the proteolytic cleavage of prolactin into an N-terminal 16kDa fragment with strong antiangiogenic and pro-apoptotic features. We hypothesize that prolactin and its cleavage products play a role in the pathopysiology of ECMO-related mortality. Methods and Materials Analysis of the kinetics of serum markers related to cardiac function, inflammation and prolactin in patients (n=47) before ECMO implantation, on defined time points during ECMOsupport and after ECMO explantation. Patients undergoing cardiac surgery (n=20) and healthy people (n=10) served as controls. Results were compared to kinetics of prolactin levels of patients requiring LVAD-implantation (n=5), after cardiac resuscitation (n=10), during sepsis (n=5) and patients with longterm LVAD-support with either stable (n= 25) or complicated (n=10) clinical course. Results Baseline prolactin levels were similar between ECMO-patients and control groups. Prolactin levels increased markedly after connection to ECMO, (from a mean of 20μg/l to a mean of 42μg/l in 24h), further increased during ECMO support (up to 174μg/l) and decreased after weaning from ECMO. Patients who died while being connected to ECMO or after weaning, developed prolactin levels which were significantly higher (mean 73μg/l ± 48 μg/l) compared to ECMO patients who survived (mean 28μg/l, ± 11 μg/l, p Conclusions Elevated prolactin levels are associated with a poor outcome in patients receiving ECMO-support, suggesting that prolactin could be used for risk stratification in these patients. In addition, with the potential adverse effect of 16kD prolactin on the endothelium, it may also be involved in the pathophysiology of ECMO-related mortality.

Heart disease can be caused by many different insults and injuries and often terminates in death from heart failure, an inability of the heart muscle to adequately supply blood to the body. A major aim in today's research is to define novel therapeutic strategies to limit myocardial damage and to enhance the ability of the heart muscle to regenerate and to heal itself. The anti-cancer drug doxorubicin (DOX) is used to treat a broad range of cancers but is limited in its use by severe side effects, most notably heart failure [1]. STAT3 is an important factor that promotes survival and migration of many tumor cell types and its pharmacological blockade hold good promise as a novel anti-cancer strategy [2, 3]. However, likewise to DOX, blocking STAT3 in the heart has been associated with heart failure in various experimental models [4, 5]. In our latest work, we found that low-doses of erythropoietin (EPO), a cytokine with multiple functions best known for its role to control the production of red blood cells, reduces the risk of heart failure mediated by DOX treatment or STAT3 blockade [6]. Our findings suggest that treatment with low-doses (hematocrit-inactive) synthetic EPO (CERA) during DOX treatment or STAT3 blockade is able to preserve the ability of cardiac progenitor cells to repair the vasculature of the heart and thereby to attenuate myocardial injury [6]. Recent studies have shown that the heart possesses intrinsic cardiac stem or progenitor cells that can contribute to regeneration and healing during disease and aging [7]. Little is known about the molecules and pathways that regulate stem cell proliferation and differentiation. In order to obtain a better understanding how the cardiac microenvironment impacts on the endogenous regeneration system of the adult heart, we studied phenotypic alterations of resident cardiac progenitor cells (identified by the expression of Sca-1) in heart failure as a consequence of DOX treatment or genetic ablation of STAT3 in cardiac muscle cells. We observed that in both heart failure models, cardiac progenitor cells displayed an impaired ability to form new blood vessels essential for oxygen delivery to the heart muscle[6]. Both models also exhibited reduced production of EPO in the heart muscle[6]. Interestingly, we found that cardiac progenitor cells bind EPO within the cardiac microenvironment, a feature that seems to be required to maintain the ability to differentiate into new blood vessels[6]. Importantly, administration of the synthetic EPO derivative CERA at a low-dosage (hematocrit-inactive) restored or preserved the endothelial differentiation potential of cardiac progenitor cells in mice with depleted STAT3 or in mice obtaining DOX treatment. Moreover, CERA preserved heart blood vessels and cardiac function in both mouse models[6]. Our data imply that short-term EPO administration at low-doses seems an attractive avenue to pursue for protecting the heart against chemotherapy-induced failure and might even have broader applications in cardiac regeneration[6]. However, further evaluation of EPO in tumor research is required, especially with regard to its effect on tumor growth and anti-tumor treatment strategies.

Besides its role as a transcription factor, Signal transducer and activator of transcription 3 (Stat3) is present in mitochondria and regulates respiration (Science 2009;323:793-7). Furthermore, Stat3-deficient mice (Stat3-CKO) develop peripartum cardiomyopathy caused by oxidative stress.To determine the role of Stat3 in regulating redox state and reactive oxygen species (ROS) production in cardiac mitochondria, myocytes from mice with cardiac-specific deletion of Stat3 (Stat3-CKO) and wild-type littermates (WT) were field-stimulated at 0.5 Hz and then exposed to a transition in workload (5 Hz, isoproterenol 30 nM). Redox-states of NAD(P)H/NAD(P)+ and FADH2/FAD+ (autofluorescence) were similar in WT and Stat3-CKO at baseline (∼65% reduced, respectively) and transiently oxidized upon increased workload (to ∼53% after 3 min), suggesting similar increases in ADP-induced respiration. Mitochondrial membrane potential (ΔΨm; TMRM) was maintained during the transition in both groups. While superoxide production (.O2-; MitoSox) remained stable in WT myocytes after elevated workload, it increased ∼4-fold during the first minute after the transition in Stat3-CKO, but normalized thereafter. To test whether this affects the long-term response to β-adrenergic stimulation, Stat3-CKO and WT mice were treated with isoproterenol for 24h in vivo (osmotic mini-pumps). After this treatment, left ventricular function was maintained in vivo, but NAD(P)H/NAD(P)+ and FADH2/FAD+ redox states were substantially oxidized in isolated myocytes at baseline (∼35% reduced, respectively). After increasing workload, NAD(P)H/NAD(P)+ and FADH2/FAD+ redox states were reduced to ∼55% in WT, but further oxidized in Stat3-CKO myocytes (to 28%; p

Peripartum cardiomyopathy (PPCM) is a rare disease of unclear etiology with a frequent poor outcome, despite optimal medical therapy. Recent experimental data implicate a causal role of prolactin. We report a patient with PPCM who responded well to treatment with Bromocriptine in addition to standard therapy of heart failure.

In the common housefly, the presence or absence of a male-determining factor, M, is responsible for sex determination. In different strains, M has been found on the Y, on the X, or on any of the five autosomes. By analyzing a Y-autosomal translocation and a ring-shaped, truncated Y chromosome, we could show that M on the Y consists of at least two regions with M activity: One of them can be assigned to the short arm of the Y chromosome (MYS), which is largely C-banding negative, the other region lies on the C-banding positive long arm of the Y, including the centromeric part (MYL). Each region alone behaves as a hypomorphic M factor, causing many carriers to develop as intersexes of the mosaic type instead of as males. When introduced into the female germ line by transplantation of progenitor germ cells (pole cells), the MYS shows an almost complete maternal effect that predetermines 96% of the genotypic female (NoM) animals to develop as males. In contrast, the MYL has largely lost its maternal effect, and most of the NoM animals develop as females. Increasing the amount of product made by either of the two hypomorphic M factors (by combining the MYS and MYL or two MYS) leads to complete male development in almost every case. We thus assume that the Y chromosome carries at least two copies of M, and that these are functionally equivalent.