Rapid changes of mRNA expressions of cardiac ion channels affected by Torsadogenic drugs influence susceptibility of rat hearts to arrhythmias induced by Beta-Adrenergic stimulation.

Drug-induced long QT syndrome (LQTS) and Torsades de Pointes (TdP) are serious concerns in drug development. Although rats are a useful scientific tool, their hearts, unlike larger species, usually do not respond to torsadogenic drugs. Consequently, their resistance to drug-induced arrhythmias is poorly understood. Here, we challenged rats with rapid delayed rectifier current (Ikr)-inhibiting antibiotic clarithromycin (CLA), loop diuretic furosemide (FUR) or their combination (CLA + FUR), and examined functional and molecular abnormalities after stimulation with isoproterenol. Clarithromycin and furosemide were administered orally at 12-h intervals for 7 days. To evaluate electrical instability, electrocardiography (ECG) was recorded either in vivo or ex vivo using the Langendorff-perfused heart method under basal conditions and subsequently under beta-adrenergic stimulation. Gene expression was measured using real-time quantitative PCR in left ventricular tissue. Indeed, FUR and CLA + FUR rats exhibited hypokalemia. CLA and CLA + FUR treatment resulted in drug-induced LQTS and even an episode of TdP in one CLA + FUR rat. The combined treatment dysregulated gene expression of several ion channels subunits, including KCNQ1, calcium channels and Na+/K + -ATPase subunits, while both monotherapies had no impact. The rat with recorded TdP exhibited differences in the expression of ion channel genes compared to the rest of rats within the CLA + FUR group. The ECG changes were not detected in isolated perfused hearts. Hence, we report rapid orchestration of ion channel reprogramming of hearts with QT prolongation induced by simultaneous administration of clarithromycin and furosemide in rats, which may account for their ability to avoid arrhythmias triggered by beta-adrenergic stimulation.

The DNA Damage Response Regulates RAG1/2 Expression in Pre-B Cells through ATM-FOXO1 Signaling.

The recombination activating gene (RAG) 1 and RAG2 protein complex introduces DNA breaks at Tcr and Ig gene segments that are required for V(D)J recombination in developing lymphocytes. Proper regulation of RAG1/2 expression safeguards the ordered assembly of Ag receptors and the development of lymphocytes, while minimizing the risk for collateral damage. The ataxia telangiectasia mutated (ATM) kinase is involved in the repair of RAG1/2-mediated DNA breaks and prevents their propagation. The simultaneous occurrence of RAG1/2-dependent and -independent DNA breaks in developing lymphocytes exposed to genotoxic stress increases the risk for aberrant recombinations. In this study, we assessed the effect of genotoxic stress on RAG1/2 expression in pre-B cells and show that activation of the DNA damage response resulted in the rapid ATM-dependent downregulation of RAG1/2 mRNA and protein expression. We show that DNA damage led to the loss of FOXO1 binding to the enhancer region of the RAG1/2 locus (Erag) and provoked FOXO1 cleavage. We also show that DNA damage caused by RAG1/2 activity in pre-B cells was able to downmodulate RAG1/2 expression and activity, confirming the existence of a negative feedback regulatory mechanism. Our data suggest that pre-B cells are endowed with a protective mechanism that reduces the risk for aberrant recombinations and chromosomal translocations when exposed to DNA damage, involving the ATM-dependent regulation of FOXO1 binding to the Erag enhancer region.

NF-κB and AKT signaling prevent DNA damage in transformed pre-B cells by suppressing RAG1/2 expression and activity.

In developing lymphocytes, expression and activity of the recombination activation gene protein 1 (RAG1) and RAG2 endonuclease complex is tightly regulated to ensure ordered recombination of the immunoglobulin genes and to avoid genomic instability. Aberrant RAG activity has been implicated in the generation of secondary genetic events in human B-cell acute lymphoblastic leukemias (B-ALLs), illustrating the oncogenic potential of the RAG complex. Several layers of regulation prevent collateral genomic DNA damage by restricting RAG activity to the G1 phase of the cell cycle. In this study, we show a novel pathway that suppresses RAG expression in cycling-transformed mouse pre-B cells and human pre-B B-ALL cells that involves the negative regulation of FOXO1 by nuclear factor κB (NF-κB). Inhibition of NF-κB in cycling pre-B cells resulted in upregulation of RAG expression and recombination activity, which provoked RAG-dependent DNA damage. In agreement, we observe a negative correlation between NF-κB activity and the expression of RAG1, RAG2, and TdT in B-ALL patients. Our data suggest that targeting NF-κB in B-ALL increases the risk of RAG-dependent genomic instability.

Perinatally administered losartan augments renal ACE2 expression but not cardiac or renal Mas receptor in spontaneously hypertensive rats.

Since the identification of the alternative angiotensin converting enzyme (ACE)2/Ang-(1-7)/Mas receptor axis, renin-angiotensin system (RAS) is a new complex target for a pharmacological intervention. We investigated the expression of RAS components in the heart and kidney during the development of hypertension and its perinatal treatment with losartan in young spontaneously hypertensive rats (SHR). Expressions of RAS genes were studied by the RT-PCR in the left ventricle and kidney of rats: normotensive Wistar, untreated SHR, SHR treated with losartan since perinatal period until week 9 of age (20 mg/kg/day) and SHR treated with losartan only until week 4 of age and discontinued until week 9. In the hypertrophied left ventricle of SHR, cardiac expressions of Ace and Mas were decreased while those of AT1 receptor (Agtr1a) and Ace2 were unchanged. Continuous losartan administration reduced LV weight (0.43 ± 0.02; P < 0.05 versus SHR) but did not influence altered cardiac RAS expression. Increased blood pressure in SHR (149 ± 2 in SHR versus 109 ± 2 mmHg in Wistar; P < 0.05) was associated with a lower renal expressions of renin, Agtr1a and Mas and with an increase in ACE2. Continuous losartan administration lowered blood pressure to control levels (105 ± 3 mmHg; P < 0.05 versus SHR), however, only renal renin and ACE2 were significantly up-regulated (for both P < 0.05 versus SHR). Conclusively, prevention of hypertension and LV hypertrophy development by losartan was unrelated to cardiac or renal expression of Mas. Increased renal Ace2, and its further increase by losartan suggests the influence of locally generated Ang-(1-7) in organ response to the developing hypertension in SHRs.

Enalaprilat increases PPARß/δ expression, without influence on PPARα and PPARγ, and modulate cardiac function in sub-acute model of daunorubicin-induced cardiomyopathy.

Anthracycline therapy is limited by a cardiotoxicity that may eventually lead to chronic heart failure which is thought to be prevented by ACE inhibitors (ACEi). However, the protective effect of ACEi in early stages of this specific injury remains elusive. Activated nuclear transcription factors peroxisome proliferator-activated receptors (PPAR) regulate cellular metabolism, but their involvement in anthracycline cardiomyopathy has not been investigated yet. For this purpose, Wistar rats were administered with daunorubicin (i.p., 3 mg/kg, in 48 h intervals) or co-administered with daunorubicine and enalaprilat (i.p., 5 mg/kg in 12 h intervals). Control animals received vehicle. Left ventricular function was measured invasively under anesthesia. Cell-shortening was measured by videomicroscopy in isolated cardiomyocytes. Expression of PPARs mRNA in cardiac tissue was measured by Real-Time PCR. Although the hemodynamic parameters of daunorubicin-treated rats remained altered upon ACEi co-administration, ACEi normalized daunorubicin-induced QT prolongation. On cellular level, ACEi normalized altered basal and isoproterenol-stimulated cardiac cell shortening in daunorubicine-treated group. Moreover, anthracycline administration significantly up-regulated heart PPARα mRNA and its expression remained increased after ACEi co-administration. On the other hand, the expression of cardiac PPARß/δ was not altered in anthracycline-treated animals, whereas co-administration of ACEi increased its expression. Conclusively, effect of ACEi can be already detected in sub-acute phase of anthracycline-induced cardiotoxicity. Altered expression of heart PPARs may suggest these nuclear receptors as a novel target in anthracycline cardiomyopathy.

Differential effects of TNF (TNFSF2) and IFN-γ on intestinal epithelial cell morphogenesis and barrier function in three-dimensional culture.

BACKGROUND: The cytokines TNF (TNFSF2) and IFNγ are important mediators of inflammatory bowel diseases and contribute to enhanced intestinal epithelial permeability by stimulating apoptosis and/or disrupting tight junctions. Apoptosis and tight junctions are also important for epithelial tissue morphogenesis, but the effect of TNF and IFNγ on the process of intestinal epithelial morphogenesis is unknown. METHODS/PRINCIPAL FINDINGS: We have employed a three-dimensional cell culture system, reproducing in vivo-like multicellular organization of intestinal epithelial cells, to study the effect of TNF on intestinal epithelial morphogenesis and permeability. We show that human intestinal epithelial cells in three-dimensional culture assembled into luminal spheres consisting of a single layer of cells with structural, internal, and planar cell polarity. Exposure of preformed luminal spheres to TNF or IFNγ enhanced paracellular permeability, but via distinctive mechanisms. Thus, while both TNF and IFNγ, albeit in a distinguishable manner, induced the displacement of selected tight junction proteins, only TNF increased paracellular permeability via caspase-driven apoptosis and cell shedding. Infliximab and adalumimab inhibited these effects of TNF. Moreover, we demonstrate that TNF via its stimulatory effect on apoptosis fundamentally alters the process of intestinal epithelial morphogenesis, which contributes to the de novo generation of intestinal epithelial monolayers with increased permeability. Also IFNγ contributes to the de novo formation of monolayers with increased permeability, but in a manner that does not involve apoptosis. CONCLUSIONS: Our study provides an optimized 3D model system for the integrated analysis of (real-time) intestinal epithelial paracellular permeability and morphogenesis, and reveals apoptosis as a pivotal mechanism underlying the enhanced permeability and altered morphogenesis in response to TNF, but not IFNγ.

HSPB1, HSPB6, HSPB7 and HSPB8 protect against RhoA GTPase-induced remodeling in tachypaced atrial myocytes.

BACKGROUND: We previously demonstrated the small heat shock protein, HSPB1, to prevent tachycardia remodeling in in vitro and in vivo models for Atrial Fibrillation (AF). To gain insight into its mechanism of action, we examined the protective effect of all 10 members of the HSPB family on tachycardia remodeling. Furthermore, modulating effects of HSPB on RhoA GTPase activity and F-actin stress fiber formation were examined, as this pathway was found of prime importance in tachycardia remodeling events and the initiation of AF. METHODS AND RESULTS: Tachypacing (4 Hz) of HL-1 atrial myocytes significantly and progressively reduced the amplitude of Ca²âº transients (CaT). In addition to HSPB1, also overexpression of HSPB6, HSPB7 and HSPB8 protected against tachypacing-induced CaT reduction. The protective effect was independent of HSPB1. Moreover, tachypacing induced RhoA GTPase activity and caused F-actin stress fiber formation. The ROCK inhibitor Y27632 significantly prevented tachypacing-induced F-actin formation and CaT reductions, showing that RhoA activation is required for remodeling. Although all protective HSPB members prevented the formation of F-actin stress fibers, their mode of action differs. Whilst HSPB1, HSPB6 and HSPB7 acted via direct prevention of F-actin formation, HSPB8-protection was mediated via inhibition of RhoA GTPase activity. CONCLUSION: Overexpression of HSPB1, as well as HSPB6, HSPB7 and HSPB8 independently protect against tachycardia remodeling by attenuation of the RhoA GTPase pathway at different levels. The cardioprotective role for multiple HSPB members indicate a possible therapeutic benefit of compounds able to boost the expression of single or multiple members of the HSPB family.

Effects of different small HSPB members on contractile dysfunction and structural changes in a Drosophila melanogaster model for Atrial Fibrillation.

The most common clinical tachycardia, Atrial Fibrillation (AF), is a progressive disease, caused by cardiomyocyte remodeling, which finally results in contractile dysfunction and AF persistence. Recently, we identified a protective role of heat shock proteins (HSPs), especially the small HSPB1 member, against tachycardia remodeling in experimental AF models. Our understanding of tachycardia remodeling and anti-remodeling drugs is currently hampered by the lack of suitable (genetic) manipulatable in vivo models for rapid screening of key targets in remodeling. We hypothesized that Drosophila melanogaster can be exploited to study tachycardia remodeling and protective effects of HSPs by drug treatments or by utilizing genetically manipulated small HSP-overexpressing strains. Tachypacing of Drosophila pupae resulted in gradual and significant cardiomyocyte remodeling, demonstrated by reduced contraction rate, increase in arrhythmic episodes and reduction in heart wall shortening, compared to normal paced pupae. Heat shock, or pre-treatment with HSP-inducers GGA and BGP-15, resulted in endogenous HSP overexpression and protection against tachycardia remodeling. DmHSP23 overexpressing Drosophilas were protected against tachycardia remodeling, in contrast to overexpression of other small HSPs (DmHSP27, DmHSP67Bc, DmCG4461, DmCG7409, and DmCG14207). (Ultra)structural evaluation of the tachypaced heart wall revealed loss of sarcomeres and mitochondrial damage which were absent in tachypaced DmHSP23 overexpressing Drosophila. In addition, tachypacing induced a significant increase in calpain activity, which was prevented in tachypaced Drosophila overexpressing DmHSP23. Tachypacing of Drosophila resulted in cardiomyocyte remodeling, which was prevented by general HSP-inducing treatments and overexpression of a single small HSP, DmHSP23. Thus, tachypaced D. melanogaster can be used as an in vivo model system for rapid identification of novel targets to combat AF associated cardiomyocyte remodeling.

Enalapril decreases cardiac mass and fetal gene expression without affecting the expression of endothelin-1, transforming growth factor ß-1, or cardiotrophin-1 in the healthy normotensive rat.

Angiotensin II can induce cardiac hypertrophy by stimulating the release of growth factors. ACE inhibitors reduce angiotensin II levels and cardiac hypertrophy, but their effects on the healthy heart are largely unexplored. We hypothesized that ACE inhibition decreases left ventricular mass in normotensive animals and that this is associated with altered expression of cardiac fetal genes, growth factors, and endothelial nitric oxide synthase (eNOS). Wistar rats (n = 7 per group) were orally administered with enalapril twice daily for a total daily dose of 5 mg·kg(-1)·d(-1) (ENAP5) or 15 mg·kg(-1)·d(-1) (ENAP15) or vehicle. Systolic blood pressure was measured by the tail-cuff method. Left ventricular expression of cardiac myosin heavy chain-α (MYH6) and -ß (MYH7), atrial natriuretic peptide (ANP), endothelin-1 (ET-1), transforming growth factor ß-1 (TGFß-1), cardiotrophin-1 (CT-1), and renal renin were examined by real-time PCR, and eNOS using Western blot. Blood pressure was decreased only in ENAP15 animals (p < 0.05 vs. Control), whereas left ventricular mass decreased after both doses of enalapril (p < 0.05 vs. Control). MYH7 and ANP were reduced in ENAP15, while no changes in ET-1, TGFß-1, CT-1, and MYH6 mRNA or eNOS protein were observed. Renal renin dose-dependently increased after enalapril treatment. Enalapril significantly decreased left ventricular mass even after 1 week treatment in the normotensive rat. This was associated with a decreased expression of the fetal genes MYH7 and ANP, but not expression of ET-1, CT-1, or TGFß-1.