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AimsIn atrial fibrillation, increased function of the Na+/Ca2+-exchanger (NCX) is one among several electrical remodeling mechanisms.Methods/resultsUsing the patch-clamp- and Ca2+ imaging-methods, we investigated atrial myocytes from NCX-homozygous-overexpressor (OE)- and heterozygous-knockout (KO)-mice and their corresponding wildtypes (WTOE; WTKO). NCX mediated Ca2+ extrusion capacity was reduced in KO and increased in OE. There was no evidence for structural or molecular remodeling. During a proarrhythmic pacing-protocol, the number of low amplitude delayed afterdepolarizations (DADs) was unaltered in OE vs. WTOE and KO vs. WTKO. However, DADs triggered full spontaneous action potentials (sAP) significantly more often in OE vs. WTOE (ratio sAP/DAD: OE:0.18±0.05; WTOE:0.02±0.02; p

Background: Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme that controls Ca 2+ homeostasis and contractility of the heart via dephosphorylation of regulatory proteins. In some genetically modified mouse models with increased arrhythmogenicity, a reduced expression of the regulatory subunit B56α of PP2A was found as a concomitant effect. Whether there is a general correlation between the abundance of B56α and the promotion of cardiac arrhythmogenesis remains unclear., Methods: The aim of this study was therefore to investigate the role of PP2A-B56α in the propensity for arrhythmic activity in the heart. The experimental analysis of this question has been addressed by using a mouse model with deletion of the PP2A-B56α gene, PPP2R5A (KO), in comparison to wild-type animals (WT). Evidence for arrhythmogenicity was investigated in whole animal, isolated heart and cardiomyocytes by ECG, recording of monophasic action potential (MAP) induced by programmed electrical stimulation (PES), measurement of Ca 2+ transients under increased pacing frequencies and determination of total K + channel currents ( I K )., Results: ECG measurements showed a prolongation of QT time in KO vs. WT. KO mice exhibited a higher rate of premature ventricular contractions in the ECG. MAP measurements in Langendorff-perfused KO hearts showed increased episodes of ventricular tachyarrhythmia induced by PES. However, the KO hearts showed values for MAP duration that were similar to those in WT hearts. In contrast, KO showed more myocardial cells with spontaneous arrhythmogenic Ca 2+ transient events compared to WT. The whole-cell patch-clamp technique applied to ventricular cardiomyocytes revealed comparable peak potassium channel current densities between KO and WT., Conclusion: These findings support the assumption that a decrease or even the loss of PP2A-B56α leads to an increased propensity of triggered arrhythmias. This could be based on the increased spontaneous Ca 2+ tansients observed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Pluteanu, Glaser, Massing, Schulte and Kirchhefer.)

Viral myocarditis is pathologically associated with RNA viruses such as coxsackievirus B3 (CVB3), or more recently, with SARS-CoV-2, but despite intensive research, clinically proven treatment is limited. Here, by use of a transgenic mouse strain (TG) containing a CVB3ΔVP0 genome we unravel virus-mediated cardiac pathophysiological processes in vivo and in vitro. Cardiac function, pathologic ECG alterations, calcium homeostasis, intracellular organization and gene expression were significantly altered in transgenic mice. A marked alteration of mitochondrial structure and gene expression indicates mitochondrial impairment potentially contributing to cardiac contractile dysfunction. An extended picture on viral myocarditis emerges that may help to develop new treatment strategies and to counter cardiac failure.

Aims: Na + /Ca 2+ exchanger (NCX) upregulation in cardiac diseases like heart failure promotes as an independent proarrhythmic factor early and delayed afterdepolarizations (EADs/DADs) on the single cell level. Consequently, NCX inhibition protects against EADs and DADs in isolated cardiomyocytes. We here investigate, whether these promising cellular in vitro findings likewise apply to an in vivo setup., Methods/results: Programmed ventricular stimulation (PVS) and isoproterenol were applied to a murine heterozygous NCX-knockout model (KO) to investigate ventricular arrhythmia initiation and perpetuation compared to wild-type (WT). KO displayed a reduced susceptibility towards isoproterenol-induced premature ventricular complexes. During PVS, initiation of single or double ectopic beats was similar between KO and WT. But strikingly, perpetuation of ventricular tachycardia (VT) was significantly increased in KO (animals with VT - KO: 82 %; WT: 47 %; p = 0.0122 / median number of VTs - KO: 4.5 (1.0, 6.25); WT: 0.0 (0.0, 4.0); p = 0.0039). The median VT duration was prolonged in KO (in s; KO: 0.38 (0.19, 0.96); WT: 0.0 (0.0, 0.60); p = 0.0239). The ventricular refractory period (VRP) was shortened in KO (in ms; KO: 15.1 ± 0.7; WT: 18.7 ± 0.7; p = 0.0013)., Conclusions: Not the initiation, but the perpetuation of provoked whole-heart in vivo ventricular arrhythmia was increased in KO. As a potential mechanism, we found a significantly reduced VRP, which may promote perpetuation of reentrant ventricular arrhythmia. On a translational perspective, the antiarrhythmic concept of therapeutic NCX inhibition seems to be ambivalent by protecting from initiating afterdepolarizations but favoring arrhythmia perpetuation in vivo at least in a murine model., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors. Published by Elsevier B.V.)

The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56α is the dominant isoform of the B'-family in the heart. Its role in regulating the cardiac response to β-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56α to test the functional cardiac effects in response to catecholamine administration versus corresponding WT mice. We found the decrease in basal PP2A activity in hearts of KO mice was accompanied by a counter-regulatory increase in the expression of B' subunits (β and γ) and higher phosphorylation of sarcoplasmic reticulum Ca 2+ regulatory and myofilament proteins. The higher phosphorylation levels were associated with enhanced intraventricular pressure and relaxation in catheterized KO mice. In contrast, at the cellular level, we detected depressed Ca 2+ transient and sarcomere shortening parameters in KO mice at basal conditions. Consistently, the peak amplitude of the L-type Ca 2+ current was reduced and the inactivation kinetics of I CaL were prolonged in KO cardiomyocytes. However, we show β-adrenergic stimulation resulted in a comparable peak amplitude of Ca 2+ transients and myocellular contraction between KO and WT cardiomyocytes. Therefore, we propose higher isoprenaline-induced Ca 2+ spark frequencies might facilitate the normalized Ca 2+ signaling in KO cardiomyocytes. In addition, the application of isoprenaline was associated with unchanged L-type Ca 2+ current parameters between both groups. Our data suggest an important influence of PP2A-B56α on the regulation of Ca 2+ signaling and contractility in response to β-adrenergic stimulation in the myocardium., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Prostate cancer is a common cause of death worldwide. Here, we isolated cancer stem cells (CSCs) from four adenocarcinomas of the prostate (Gleason scores from 3 + 3 up to 4 + 5). CSCs were characterized by the expression of the stem cell markers TWIST , the epithelial cell adhesion molecule ( EPCAM ), the transcription factors SNAI1 ( SNAIL ) and SNAI2 ( SLUG ) and cancer markers such as CD44 and prominin-1 (CD133). All investigated CSC populations contained a fraction highly positive for aldehyde dehydrogenase (ALDH) function and displayed robust expressions of programmed cell death 1 (PD-1) ligands. Furthermore, we investigated immunotherapeutic approaches but had no success even with the clinically used PD-1 inhibitor pembrolizumab. In addition, we studied another death-inducing pathway via interferon gamma signaling and detected high-level upregulations of human leukocyte antigen A (HLA-A) and beta 2-microglobulin (B2M) with only moderate killing efficacy. To examine further killing mechanisms in prostate cancer stem cells (PCSCs), we analyzed NF-κB signaling. Surprisingly, two patient-specific populations of PCSCs were found: one with canonical NF-κB signaling and another one with blunted NF-κB activation, which can be efficiently killed by tumor necrosis factor (TNF). Thus, culturing of PCSCs and analysis of respective NF-κB induction potency after surgery might be a powerful tool for optimizing patient-specific treatment options, such as the use of TNF-inducing chemotherapeutics and/or NF-κB inhibitors.

ICER (inducible cAMP early repressor) isoforms are transcriptional repressors encoded by the Crem (cAMP responsive element modulator) gene. They were linked to the regulation of a multitude of cellular processes and pathophysiological mechanisms. Here, we show for the first time that two independent induction patterns for CREM repressor isoforms exist in the heart, namely for ICER and smICER (small ICER), which are induced in response to β-adrenergic stimulation in a transient- and saturation-like manner, respectively. This time-shifted induction pattern, driven by two internal promoters in the Crem gene, leads to the predominant transcription of smIcer after prolonged β-adrenergic stimulation. Using an ICER knockout mouse model with preserved smICER induction, we show that the transient-like induction of Icer itself has minor effects on gene regulation, cardiac hypertrophy or contractile function in the heart. We conclude that the functions previously linked to ICER may be rather attributed to smICER, also beyond the cardiac background., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: A structural, electrical and metabolic atrial remodeling is central in the development of atrial fibrillation (AF) contributing to its initiation and perpetuation. In the heart, HDACs (histone deacetylases) control remodeling associated processes like hypertrophy, fibrosis, and energy metabolism. Here, we analyzed, whether the HDAC class I/IIa inhibitor valproic acid (VPA) is able to attenuate atrial remodeling in CREM-IbΔC-X (cAMP responsive element modulator isoform IbΔC-X) transgenic mice, a mouse model of extensive atrial remodeling with age-dependent progression from spontaneous atrial ectopy to paroxysmal and finally long-lasting AF., Methods: VPA was administered for 7 or 25 weeks to transgenic and control mice. Atria were analyzed macroscopically and using widefield and electron microscopy. Action potentials were recorded from atrial cardiomyocytes using patch-clamp technique. ECG recordings documented the onset of AF. A proteome analysis with consecutive pathway mapping identified VPA-mediated proteomic changes and related pathways., Results: VPA attenuated many components of atrial remodeling that are present in transgenic mice, animal AF models, and human AF. VPA significantly ( P<0.05) reduced atrial dilatation, cardiomyocyte enlargement, atrial fibrosis, and the disorganization of myocyte's ultrastructure. It significantly reduced the occurrence of atrial thrombi, reversed action potential alterations, and finally delayed the onset of AF by 4 to 8 weeks. Increased histone H4-acetylation in atria from VPA-treated transgenic mice verified effective in vivo HDAC inhibition. Cardiomyocyte-specific genetic inactivation of HDAC2 in transgenic mice attenuated the ultrastructural disorganization of myocytes comparable to VPA. Finally, VPA restrained dysregulation of proteins in transgenic mice that are involved in a multitude of AF relevant pathways like oxidative phosphorylation or RhoA (Ras homolog gene family, member A) signaling and disease functions like cardiac fibrosis and apoptosis of muscle cells., Conclusions: Our results suggest that VPA, clinically available, well-tolerated, and prescribed to many patients for years, has the therapeutic potential to delay the development of atrial remodeling and the onset of AF in patients at risk.

Administration of digitalis in heart failure (HF) increases quality of life but does not carry a prognostic benefit. Digitalis is an indirect inhibitor of the Na + /Ca 2+ exchanger (NCX), which is overexpressed in HF. We therefore used the cardiac glycoside ouabain in Ca 2+ imaging experiments and patch-clamp experiments in isolated ventricular myocytes from nonfailing transgenic NCX overexpressor mice (OE). In field-stimulated myocytes, ouabain (1-100 μm) increased the amplitude of the Ca 2+ transient in OE and wild-type (WT) similarly. Ouabain-mediated spontaneous Ca 2+ -activity was significantly more pronounced in OE compared to WT myocytes at higher concentrations (100 μm). Also, at very high concentrations (1000 μm) of ouabain, the number of cells with hypercontraction leading to cell death was higher in OE. Ouabain (10 μm) shortened the action potential duration in both genotypes. Our findings suggest that the proarrhythmic but not the inotropic effects of cardiac glycosides are enhanced by increased NCX expression. This may offer an explanation for the observed lack of prognostic benefit but increased quality of life in HF, which is accompanied by NCX upregulation., (© 2018 Société Française de Pharmacologie et de Thérapeutique.)

Rationale: A higher expression/activity of type 1 serine/threonine protein phosphatase 1 (PP1) may contribute to dephosphorylation of cardiac regulatory proteins triggering the development of heart failure., Objective: Here, we tested the putatively protective effects of PP1 inhibitor-2 (I 2 ) overexpression using a heart failure model induced by chronic β-adrenergic stimulation., Methods and Results: Transgenic (TG) and wild-type (WT) mice were subjected to isoprenaline (ISO) or isotonic NaCl solution supplied via osmotic minipumps for 7 days. I 2 overexpression was associated with a depressed PP1 activity. Basal contractility was unchanged in catheterized mice and isolated cardiomyocytes between TG NaCl and WT NaCl . TG ISO mice exhibited more fibrosis and a higher expression of hypertrophy marker proteins as compared to WT ISO . After acute administration of ISO, the contractile response was accompanied by a higher sensitivity in TG ISO as compared to WT ISO . In contrast to basal contractility, the peak amplitude of [Ca] i and SR Ca load were reduced in TG NaCl as compared to WT NaCl . These effects were normalized to WT levels after chronic ISO stimulation. Cardiomyocyte relaxation and [Ca] i decay kinetics were hastened in TG ISO as compared to WT ISO , which can be explained by a higher phospholamban phosphorylation at Ser 16 . Chronic catecholamine stimulation was followed by an enhanced expression of GSK3β, whereas the phosphorylation at Ser 9 was lower in TG as compared to the corresponding WT group. This resulted in a higher I 2 phosphorylation that may reactivate PP1., Conclusion: Our findings suggest that the basal desensitization of β-adrenergic signaling and the depressed Ca handling in TG by inhibition of PP1 is restored by a GSK3β-dependent phosphorylation of I 2 ., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Principal mechanisms of arrhythmia have been derived from ventricular but not atrial cardiomyocytes of animal models despite higher prevalence of atrial arrhythmia (e.g., atrial fibrillation). Due to significant ultrastructural and functional differences, a simple transfer of ventricular proneness toward arrhythmia to atrial arrhythmia is critical. The use of murine models in arrhythmia research is widespread, despite known translational limitations. We here directly compare atrial and ventricular mechanisms of arrhythmia to identify critical differences that should be considered in murine models for development of antiarrhythmic strategies for atrial arrhythmia. Methods and Results: Isolated murine atrial and ventricular myocytes were analyzed by wide field microscopy and subjected to a proarrhythmic protocol during patch-clamp experiments. As expected, the spindle shaped atrial myocytes showed decreased cell area and membrane capacitance compared to the rectangular shaped ventricular myocytes. Though delayed afterdepolarizations (DADs) could be evoked in a similar fraction of both cell types (80% of cells each), these led significantly more often to the occurrence of spontaneous action potentials (sAPs) in ventricular myocytes. Interestingly, numerous early afterdepolarizations (EADs) were observed in the majority of ventricular myocytes, but there was no EAD in any atrial myocyte (EADs per cell; atrial myocytes: 0 ± 0; n = 25/12 animals; ventricular myocytes: 1.5 [0-43]; n = 20/12 animals; p < 0.05). At the same time, the action potential duration to 90% decay (APD 90 ) was unaltered and the APD 50 even increased in atrial versus ventricular myocytes. However, the depolarizing L-type Ca 2+ current (I Ca ) and Na + /Ca 2+ -exchanger inward current (I NCX ) were significantly smaller in atrial versus ventricular myocytes. Conclusion: In mice, atrial myocytes exhibit a substantially distinct occurrence of proarrhythmic afterdepolarizations compared to ventricular myocytes, since they are in a similar manner susceptible to DADs but interestingly seem to be protected against EADs and show less sAPs. Key factors in the generation of EADs like I Ca and I NCX were significantly reduced in atrial versus ventricular myocytes, which may offer a mechanistic explanation for the observed protection against EADs. These findings may be of relevance for current studies on atrial level in murine models to develop targeted strategies for the treatment of atrial arrhythmia.

Understanding molecular mechanisms involved in atrial tissue remodeling and arrhythmogenesis in atrial fibrillation (AF) is essential for developing specific therapeutic approaches. Two-pore-domain potassium (K 2P ) channels modulate cellular excitability, and TASK-1 (K 2P 3.1) currents were recently shown to alter atrial action potential duration in AF and heart failure (HF). Finding animal models of AF that closely resemble pathophysiological alterations in human is a challenging task. This study aimed to analyze murine cardiac expression patterns of K 2P channels and to assess modulation of K 2P channel expression in murine models of AF and HF. Expression of cardiac K 2P channels was quantified by real-time qPCR and immunoblot in mouse models of AF [cAMP-response element modulator (CREM)-IbΔC-X transgenic animals] or HF (cardiac dysfunction induced by transverse aortic constriction, TAC). Cloned murine, human, and porcine TASK-1 channels were heterologously expressed in Xenopus laevis oocytes. Two-electrode voltage clamp experiments were used for functional characterization. In murine models, among members of the K 2P channel family, TASK-1 expression displayed highest levels in both atrial and ventricular tissue samples. Furthermore, K 2P 2.1, K 2P 5.1, and K 2P 6.1 showed significant expression levels. In CREM-transgenic mice, atrial expression of TASK-1 was significantly reduced in comparison with wild-type animals. In a murine model of TAC-induced pressure overload, ventricular TASK-1 expression remained unchanged, while atrial TASK-1 levels were significantly downregulated. When heterologously expressed in Xenopus oocytes, currents of murine, porcine, and human TASK-1 displayed similar characteristics. TASK-1 channels display robust cardiac expression in mice. Murine, porcine, and human TASK-1 channels share functional similarities. Dysregulation of atrial TASK-1 expression in murine AF and HF models suggests a mechanistic contribution to arrhythmogenesis.

Background: The molecular pathomechanisms underlying atrial thrombogenesis are multifactorial and still require detailed investigations. Transgenic mice with cardiomyocyte-directed expression of the transcriptional repressor CREM-IbΔC-X (CREM-TG) represent an experimental model of atrial fibrillation (AF) that shows a gradual, age-dependent progression from atrial ectopy to persistent AF. Importantly, this model develops biatrial thrombi. The molecular characteristics related to the thrombogenesis in CREM-TG mice have not been studied, yet., Methods: The inflammatory and prothrombotic state was evaluated at the transcriptional (qRT-PCR) and protein level in the left (LA) and right atria (RA) from CREM-TG mice at the age of 20weeks and compared to wild-type controls. Moreover, histological analyses of atrial thrombi were performed., Results: The endocardial dysfunction was mirrored by diminished levels of eNOS-mRNA in both atria (RA: 0.79±0.04, LA: 0.72±0.06; each P<0.05). Moreover, the PAI-1/t-PA mRNA ratio was significantly increased in both atria (RA: 3.6±0.6; P<0.01, LA: 4.0±1.0; P<0.05) indicating a high risk of thrombus formation. However, the inflammatory phenotype was more pronounced in the RA and was reflected by a significant increase in the mRNA levels encoding adhesion molecules ICAM-1 (2.1±0.2; P<0.01), VCAM-1 (2.3±0.5; P<0.05), and selectin P (3.6±0.5: P<0.05)., Conclusions: CREM-TG mice represent a valuable model for studying atrial thrombogenesis and assessing therapeutic approaches preventing embolic events in the systemic and pulmonary circulation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Two-pore-domain potassium (K 2P ) channels modulate cellular excitability. The significance of stretch-activated cardiac K 2P channels (K 2P 2.1, TREK-1, KCNK2; K 2P 4.1, TRAAK, KCNK4; K 2P 10.1, TREK-2, KCNK10) in heart disease has not been elucidated in detail. The aim of this work was to assess expression and remodeling of mechanosensitive K 2P channels in atrial fibrillation (AF) and heart failure (HF) patients in comparison to murine models. Cardiac K 2P channel levels were quantified in atrial (A) and ventricular (V) tissue obtained from patients undergoing open heart surgery. In addition, control mice and mouse models of AF (cAMP-response element modulator (CREM)-IbΔC-X transgenic animals) or HF (cardiac dysfunction induced by transverse aortic constriction, TAC) were employed. Human and murine KCNK2 displayed highest mRNA abundance among mechanosensitive members of the K 2P channel family (V > A). Disease-associated K 2P 2.1 remodeling was studied in detail. In patients with impaired left ventricular function, atrial KCNK2 (K 2P 2.1) mRNA and protein expression was significantly reduced. In AF subjects, downregulation of atrial and ventricular KCNK2 (K 2P 2.1) mRNA and protein levels was observed. AF-associated suppression of atrial Kcnk2 (K 2P 2.1) mRNA and protein was recapitulated in CREM-transgenic mice. Ventricular Kcnk2 expression was not significantly altered in mouse models of disease. In conclusion, mechanosensitive K 2P 2.1 and K 2P 10.1 K + channels are expressed throughout the heart. HF- and AF-associated downregulation of KCNK2 (K 2P 2.1) mRNA and protein levels suggest a mechanistic contribution to cardiac arrhythmogenesis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background/Objective: The cardiac Na + /Ca 2+ exchanger (NCX) has been identified as a promising target to counter arrhythmia in previous studies investigating the benefit of NCX inhibition. However, the consequences of NCX inhibition have not been investigated in the setting of altered NCX expression and function, which is essential, since major cardiac diseases (heart failure/atrial fibrillation) exhibit NCX upregulation. Thus, we here investigated the effects of the NCX inhibitor SEA0400 on the Ca 2+ transient amplitude and on proarrhythmia in homozygous NCX overexpressor (OE) and heterozygous NCX knockout (hetKO) mice compared to corresponding wild-types (WT OE /WT hetKO ). Methods/Results: Ca 2+ transients of field-stimulated isolated ventricular cardiomyocytes were recorded with fluo-4-AM or indo-1-AM. SEA0400 (1 μM) significantly reduced NCX forward mode function in all mouse lines. SEA0400 (1 μM) significantly increased the amplitude of field-stimulated Ca 2+ transients in WT OE , WT hetKO , and hetKO, but not in OE (% of basal; OE = 98.7 ± 5.0; WT OE = 137.8 ± 5.2 * ; WT hetKO = 126.3 ± 6.0 * ; hetKO = 140.6 ± 12.8 * ; * p < 0.05 vs. basal). SEA0400 (1 μM) significantly reduced the number of proarrhythmic spontaneous Ca 2+ transients (sCR) in OE, but increased it in WT OE , WT hetKO and hetKO (sCR per cell; basal/+SEA0400; OE = 12.5/3.7; WT OE = 0.2/2.4; WT hetKO = 1.3/8.8; hetKO = 0.2/5.5) and induced Ca 2+ overload with subsequent cell death in hetKO. Conclusion: The effects of SEA0400 on Ca 2+ transient amplitude and the occurrence of spontaneous Ca 2+ transients as a proxy measure for inotropy and cellular proarrhythmia depend on the NCX expression level. The antiarrhythmic effect of SEA0400 in conditions of increased NCX expression promotes the therapeutic concept of NCX inhibition in heart failure/atrial fibrillation. Conversely, in conditions of reduced NCX expression, SEA0400 suppressed the NCX function below a critical level leading to adverse Ca 2+ accumulation as reflected by an increase in Ca 2+ transient amplitude, proarrhythmia and cell death. Thus, the remaining NCX function under inhibition may be a critical factor determining the inotropic and antiarrhythmic efficacy of SEA0400.

Aims: In atrial fibrillation, increased function of the Na + /Ca 2+ -exchanger (NCX) is one among several electrical remodeling mechanisms., Methods/results: Using the patch-clamp- and Ca 2+ imaging-methods, we investigated atrial myocytes from NCX-homozygous-overexpressor (OE)- and heterozygous-knockout (KO)-mice and their corresponding wildtypes (WT OE ; WT KO ). NCX mediated Ca 2+ extrusion capacity was reduced in KO and increased in OE. There was no evidence for structural or molecular remodeling. During a proarrhythmic pacing-protocol, the number of low amplitude delayed afterdepolarizations (DADs) was unaltered in OE vs. WT OE and KO vs. WT KO . However, DADs triggered full spontaneous action potentials (sAP) significantly more often in OE vs. WT OE (ratio sAP/DAD: OE:0.18±0.05; WT OE :0.02±0.02; p<0.001). Using the same protocol, a DAD triggered an sAP by tendency less often in KO vs. WT KO (p=0.06) and significantly less often under a more aggressive proarrhythmic protocol (ratio sAP/DAD: KO:0.01±0.003; WT KO: 0.12±0.05; p=0.007). The DAD amplitude was increased in OE vs. WT OE and decreased in KO vs. WT KO . There were no differences in SR-Ca 2+ -load, the number of spontaneous Ca 2+ -release-events or IKACh/IK1., Conclusions: Atrial myocytes with increased NCX expression exhibited increased vulnerability towards sAPs while atriomyocytes with reduced NCX expression were protected. The underlying mechanism consists of a modification of the DAD-amplitude by the level of NCX-activity. Thus, although the number of spontaneous Ca 2+ -releases and therefore DADs is unaltered, the higher DAD-amplitude in OE made a transgression of the voltage-threshold of an sAP more likely. These findings indicate that the level of NCX activity could influence triggered activity in atrial myocytes independent of possible remodeling processes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Sphingosine-1-phosphate plays vital roles in cardiomyocyte physiology, myocardial ischemia-reperfusion injury, and ischemic preconditioning. The function of the cardiomyocyte sphingosine-1-phosphate receptor 1 (S1P1) in vivo is unknown., Methods and Results: Cardiomyocyte-restricted deletion of S1P1 in mice (S1P1 (α) (MHCC) (re)) resulted in progressive cardiomyopathy, compromised response to dobutamine, and premature death. Isolated cardiomyocytes from S1P1 (α) (MHCC) (re) mice revealed reduced diastolic and systolic Ca(2+) concentrations that were secondary to reduced intracellular Na(+) and caused by suppressed activity of the sarcolemmal Na(+)/H(+) exchanger NHE-1 in the absence of S1P1. This scenario was successfully reproduced in wild-type cardiomyocytes by pharmacological inhibition of S1P1 or sphingosine kinases. Furthermore, Sarcomere shortening of S1P1 (α) (MHCC) (re) cardiomyocytes was intact, but sarcomere relaxation was attenuated and Ca(2+) sensitivity increased, respectively. This went along with reduced phosphorylation of regulatory myofilament proteins such as myosin light chain 2, myosin-binding protein C, and troponin I. In addition, S1P1 mediated the inhibitory effect of exogenous sphingosine-1-phosphate on β-adrenergic-induced cardiomyocyte contractility by inhibiting the adenylate cyclase. Furthermore, ischemic precondtioning was abolished in S1P1 (α) (MHCC) (re) mice and was accompanied by defective Akt activation during preconditioning., Conclusions: Tonic S1P1 signaling by endogenous sphingosine-1-phosphate contributes to intracellular Ca(2+) homeostasis by maintaining basal NHE-1 activity and controls simultaneously myofibril Ca(2+) sensitivity through its inhibitory effect on adenylate cyclase. Cardioprotection by ischemic precondtioning depends on intact S1P1 signaling. These key findings on S1P1 functions in cardiac physiology may offer novel therapeutic approaches to cardiac diseases., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Chronic β-adrenergic stimulation is regarded as a pivotal step in the progression of heart failure which is associated with a high risk for arrhythmia. The cAMP-dependent transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) mediate transcriptional regulation in response to β-adrenergic stimulation and CREM repressor isoforms are induced after stimulation of the β-adrenoceptor. Here, we investigate whether CREM repressors contribute to the arrhythmogenic remodeling in the heart by analyzing arrhythmogenic alterations in ventricular cardiomyocytes (VCMs) from mice with transgenic expression of the CREM repressor isoform CREM-IbΔC-X (TG). Patch clamp analyses, calcium imaging, immunoblotting and real-time quantitative RT-PCR were conducted to study proarrhythmic alterations in TG VCMs vs. wild-type controls. The percentage of VCMs displaying spontaneous supra-threshold transient-like Ca(2+) releases was increased in TG accompanied by an enhanced transduction rate of sub-threshold Ca(2+) waves into these supra-threshold events. As a likely cause we discovered enhanced NCX-mediated Ca(2+) transport and NCX1 protein level in TG. An increase in I NCX and decrease in I to and its accessory channel subunit KChIP2 was associated with action potential prolongation and an increased proportion of TG VCMs showing early afterdepolarizations. Finally, ventricular extrasystoles were augmented in TG mice underlining the in vivo relevance of our findings. Transgenic expression of CREM-IbΔC-X in mouse VCMs leads to distinct arrhythmogenic alterations. Since CREM repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of CRE-dependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease.

Background: The Na(+)/Ca(2+) exchanger (NCX) has been implied to cause arrhythmias. To date, information on the role of NCX in arrhythmogenesis derived from models with increased NCX expression, hypertrophy, and heart failure. Furthermore, the exact mechanism by which NCX exerts its potentially proarrhythmic effect, ie, by promoting early afterdepolarization (EAD) or delayed afterdepolarization (DAD) or both, is unknown., Methods and Results: We investigated isolated cardiomyocytes from a murine model with heterozygous knockout of NCX (hetKO) using the patch clamp and Ca(2+) imaging techniques. Action potential duration was shorter in hetKO with IKtot not being increased. The rate of spontaneous Ca(2+) release events and the rate of DADs were unaltered; however, DADs had lower amplitude in hetKO. A DAD triggered a spontaneous action potential significantly less often in hetKO when compared with wild-type. The occurrence of EADs was also drastically reduced in hetKO. ICa activity was reduced in hetKO, an effect that was abolished in the presence of the Ca(2+) buffer BAPTA., Conclusions: Genetic suppression of NCX reduces both EADs and DADs. The following molecular mechanisms apply: (1) Although the absolute number of DADs is unaffected, an impaired translation of DADs into spontaneous action potentials results from a reduced DAD amplitude. (2) EADs are reduced in absolute number of occurrence, which is presumably a consequence of shortened action potential duration because of reduced NCX activity but also reduced ICa the latter possibly being caused by a direct modulation of Ca(2+)-dependent ICa inhibition by reduced NCX activity. This is the first study to demonstrate that genetic inhibition of NCX protects against afterdepolarizations and to investigate the underlying mechanisms., (© 2015 American Heart Association, Inc.)

Annexin A4 (AnxA4), a Ca(2+)- and phospholipid-binding protein, is up-regulated in the human failing heart. In this study, we examined the impact of AnxA4 on β-adrenoceptor (β-AR)/cAMP-dependent signal transduction. Expression of murine AnxA4 in human embryonic kidney (HEK)293 cells dose-dependently inhibited cAMP levels after direct stimulation of adenylyl cyclases (ACs) with forskolin (FSK), as determined with an exchange protein activated by cAMP-Förster resonance energy transfer (EPAC-FRET) sensor and an ELISA (control vs. +AnxA4: 1956 ± 162 vs. 1304 ± 185 fmol/µg protein; n = 8). Disruption of the anxA4 gene led to a consistent increase in intracellular cAMP levels in isolated adult mouse cardiomyocytes, with heart-directed expression of the EPAC-FRET sensor, stimulated with FSK, and as determined by ELISA, also in mouse cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO) (anxA4a(+/+) vs. anxA4a(-/-): 5.1 ± 0.3 vs. 6.7 ± 0.6 fmol/µg protein) or FSK (anxA4a(+/+) vs. anxA4a(-/-): 1891 ± 238 vs. 2796 ± 343 fmol/µg protein; n = 9-10). Coimmunoprecipitation experiments in HEK293 cells revealed a direct interaction of murine AnxA4 with human membrane-bound AC type 5 (AC5). As a functional consequence of AnxA4-mediated AC inhibition, AnxA4 inhibited the FSK-induced transcriptional activation mediated by the cAMP response element (CRE) in reporter gene studies (10-fold vs. control; n = 4 transfections) and reduced the FSK-induced phosphorylation of the CRE-binding protein (CREB) measured on Western blots (control vs. +AnxA4: 150 ± 17% vs. 105 ± 10%; n = 6) and by the use of the indicator of CREB activation caused by phosphorylation (ICAP)-FRET sensor, indicating CREB phosphorylation. Inactivation of AnxA4 in anxA4a(-/-) mice was associated with an increased cardiac response to β-AR stimulation. Together, these results suggest that AnxA4 is a novel direct negative regulator of AC5, adding a new facet to the functions of annexins., (© FASEB.)

Directed cardiac differentiation of human pluripotent stem cells (hPSCs) enables disease modeling, investigation of human cardiogenesis, as well as large-scale production of cardiomyocytes (CMs) for translational purposes. Multiple CM differentiation protocols have been developed to individually address specific requirements of these diverse applications, such as enhanced purity at a small scale or mass production at a larger scale. However, there is no universal high-efficiency procedure for generating CMs both in two-dimensional (2D) and three-dimensional (3D) culture formats, and undefined or complex media additives compromise functional analysis or cost-efficient upscaling. Using systematic combinatorial optimization, we have narrowed down the key requirements for efficient cardiac induction of hPSCs. This implied differentiation in simple serum and serum albumin-free basal media, mediated by a minimal set of signaling pathway manipulations at moderate factor concentrations. The method was applicable both to 2D and 3D culture formats as well as to independent hPSC lines. Global time-course gene expression analyses over extended time periods and in comparison with human heart tissue were used to monitor culture-induced maturation of the resulting CMs. This suggested that hPSC-CMs obtained with our procedure reach a rather stable transcriptomic state after approximately 4 weeks of culture. The underlying gene expression changes correlated well with a decline of immature characteristics as well as with a gain of structural and physiological maturation features within this time frame. These data link gene expression patterns of hPSC-CMs to functional readouts and thus define the cornerstones of culture-induced maturation., (© 2015 AlphaMed Press.)

Background: New therapeutic approaches to improve cardiac contractility without severe risk would improve the management of acute heart failure. Increasing systolic sodium influx can increase cardiac contractility, but most sodium channel activators have proarrhythmic effects that limit their clinical use. Here, we report the cardiac effects of a novel positive inotropic peptide isolated from the toxin of the Black Judean scorpion that activates neuronal tetrodotoxin-sensitive sodium channels., Methods and Results: All venoms and peptides were isolated from Black Judean Scorpions (Buthotus Hottentotta) caught in the Judean Desert. The full scorpion venom increased left ventricular function in sedated mice in vivo, prolonged ventricular repolarization, and provoked ventricular arrhythmias. An inotropic peptide (BjIP) isolated from the full venom by chromatography increased cardiac contractility but did neither provoke ventricular arrhythmias nor prolong cardiac repolarization. BjIP increased intracellular calcium in ventricular cardiomyocytes and prolonged inactivation of the cardiac sodium current. Low concentrations of tetrodotoxin (200 nmol/L) abolished the effect of BjIP on calcium transients and sodium current. BjIP did not alter the function of Nav1.5, but selectively activated the brain-type sodium channels Nav1.6 or Nav1.3 in cellular electrophysiological recordings obtained from rodent thalamic slices. Nav1.3 (SCN3A) mRNA was detected in human and mouse heart tissue., Conclusions: Our pilot experiments suggest that selective activation of tetrodotoxin-sensitive neuronal sodium channels can safely increase cardiac contractility. As such, the peptide described here may become a lead compound for a new class of positive inotropic agents., (© 2014 American Heart Association, Inc.)

Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of β-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of -5 mV, the ICa,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser(16) was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca(2+) sensitivity and increased basal contractility in TG hearts. These effects were reversed by β-adrenergic stimulation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Home (HBPM) and ambulatory (ABPM) blood pressure measurements have their advantages and disadvantages in diagnosing and managing hypertension. We studied HBPMs and ABPMs in volunteers taking part in a survey., Methods: Of 366 respondents, 270 provided a total of 5997 triplicate HBPMs (Part 1); 175 also provided data on ABPMs, of which the measurements obtained between 6 am and 10 pm were used in this study (Part 2)., Results: Part 1, When all 5997 triplicate HPPMs were considered, 1st readings tended to be significantly higher than those of the 2nd and 3rd for both, systolic and diastolic pressures, but when the consideration was restricted to the very first triplicate of each of the 270 subjects, this was true only for systolic HBPM. Part 2, The ABPMs tended to have a wider range than the corresponding HBPMs, and to be distributed towards higher values. Of the non-parametric indices of (ABPM - corresponding HBPM), (First Quartile, Median, Third Quartile and Maximim) all but the minima had positive values., Conclusions: In triplicate HBPMs, the first measurement is usually but not always the highest. Increasing the number of triplicates provided by each subject increases the chance of discriminating between measurements in the triplicates. ABPMs tended to be higher than the corresponding HBPMs.

Human congestive heart failure is accompanied by structural and electrical alterations leading to the development of an arrhythmogenic substrate. This substrate is associated with the "sudden cardiac death" due to ventricular tachycardia or ventricular fibrillation. Multiple studies link distinct transcription factors to the transcriptional regulation of genes related to the formation of an arrhythmogenic substrate. In addition to cardiac hypertrophy the up- or downregulation of ion channels, calcium-handling proteins, and proteins forming gap junctions play a pivotal role in the progression of heart failure. This review summarizes the transcriptional regulation of selected genes implicated in the formation of an arrhythmogenic substrate. In this context we provide an overview of relevant transcription factors, activating stimuli and pathways, the evidence of binding to respective elements in the promoter of target genes and the associated mRNA regulation in animal models. Finally, possible therapeutic consequences are discussed.

Background: TNF-related apoptosis inducing ligand (TRAIL) belongs to the TNF-superfamily that induces apoptotic cell death in a wide range of neoplastic cells in vivo as well as in vitro. We identified two alternative TRAIL-splice variants, i.e. TRAIL-β and TRAIL-γ that are characterized by the loss of their proapoptotic properties. Herein, we investigated the expression and the prognostic values of the TRAIL-splice variants in gastric carcinomas., Methods: Real time PCR for amplification of the TRAIL-splice variants was performed in tumour tissue specimens and corresponding normal tissues of 41 consecutive patients with gastric carcinoma. Differences on mRNA-expression levels of the TRAIL-isoforms were compared to histo-pathological variables and correlated with survival data., Results: All three TRAIL-splice variants could be detected in both non-malignant and malignant tissues, irrespective of their histological staging, grading or tumour types. However, TRAIL-β exhibited a higher expression in normal gastric tissue. The proapoptotic TRAIL-α expression was increased in gastric carcinomas when compared to TRAIL-β and TRAIL-γ. In addition, overexpression of TRAIL-γ was associated with a significant higher survival rate., Conclusions: This is the first study that investigated the expression of TRAIL-splice variants in gastric carcinoma tissue samples. Thus, we provide first data that indicate a prognostic value for TRAIL-γ overexpression in this tumour entity.

Background: The association of EpCAM expression with the progression of gastric cancer remains unclear. Here, we investigated the expression of EpCAM in gastric cancer subtypes and correlated the data to tumor cell proliferation and clinicopathologic factors., Methods: The intratumoral expression of EpCAM was assessed in 163 primary gastric cancers (61 diffuse-, 62 intestinal-, 32 mixed-type and 8 unclassified tumors) by immunohistochemistry, using the monoclonal antibody Ber-EP4. Intensity of staining was classified according the HercepTest-score using a standardized scoring system. Ki-67 was used to examine the proliferation in tumor tissue., Results: Strong EpCAM expression was observed in 77% of the tumors and in 85% of the corresponding lymph nodes. Of the primary tumors, 58% (n=74) presented a homogeneous intratumoral EpCAM expression while 42% were characterised by a heterogenous expression pattern. Tumors with high EpCAM expression at the invasive front were associated with significantly (p=0.03) higher proportion of lymph node metastases and lower median overall survival (p=0.001). Diffuse type tumors presented a significantly higher EpCAM expression at the invasion front compared with the tumor centre (p=0.036). Multivariate survival analysis identified high EpCAM expression at the invasive front as an independent prognostic factor.We observed a significant (p=0.001) correlation between high EpCAM expression and higher tumor cell proliferation., Conclusion: High EpCAM expression associates with proliferation and progression of gastric cancer, especially in the diffuse type. Considering the discontenting results of the current adjuvant concepts for gastric cancer patients, EpCAM might be target in the adjuvant therapy of this malignant disease.

Background and Methods: Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. The substrate of AF is composed of a complex interplay between structural and functional changes of the atrial myocardium often preceding the occurrence of persistent AF. However, there are only few animal models reproducing the slow progression of the AF substrate to the spontaneous occurrence of the arrhythmia. Transgenic mice (TG) with cardiomyocyte-directed expression of CREM-IbΔC-X, an isoform of transcription factor CREM, develop atrial dilatation and spontaneous-onset AF. Here we tested the hypothesis that TG mice develop an arrhythmogenic substrate preceding AF using physiological and biochemical techniques., Results: Overexpression of CREM-IbΔC-X in young TG mice (<8weeks) led to atrial dilatation combined with distension of myocardium, elongated myocytes, little fibrosis, down-regulation of connexin 40, loss of excitability with a number of depolarized myocytes, atrial ectopies and inducibility of AF. These abnormalities continuously progressed with age resulting in interatrial conduction block, increased atrial conduction heterogeneity, leaky sarcoplasmic reticulum calcium stores and the spontaneous occurrence of paroxysmal and later persistent AF. This distinct atrial remodelling was associated with a pattern of non-regulated and up-regulated marker genes of myocardial hypertrophy and fibrosis., Conclusions: Expression of CREM-IbΔC-X in TG hearts evokes abnormal growth and development of the atria preceding conduction abnormalities and altered calcium homeostasis and the development of spontaneous and persistent AF. We conclude that transcription factor CREM is an important regulator of atrial growth implicated in the development of an arrhythmogenic substrate in TG mice., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: There is increasing evidence that serotonin (5-hydroxytryptamine [5-HT]) and distinct 5-HT receptors are involved in the pathogenesis of systemic sclerosis. The aim of this study was to test the hypothesis that tropisetron, a routinely used antiemetic agent previously characterized as a 5-HT(3/4) receptor-modulating agent, can directly affect collagen synthesis in vitro and attenuate experimentally induced fibrosis in vivo., Methods: Functional in vitro studies were performed using human dermal fibroblasts (HDFs). Signal transduction studies included immunofluorescence analysis, Western immunoblotting, promoter reporter assays, cAMP/Ca(2+) measurements, and use of pharmacologic activators and inhibitors. Gene silencing was performed using small interfering RNA. Putative receptors of tropisetron were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. The murine model of bleomycin-induced scleroderma was used to assess the antifibrogenic and antifibrotic effects of tropisetron in vivo. Collagen expression in vitro, ex vivo, and in situ was determined by real-time RT-PCR analysis, Western immunoblotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunohistochemical analysis., Results: Tropisetron suppressed collagen synthesis induced by transforming growth factor β1 (TGFβ1). This effect was independent of 5-HT(3/4) receptor but was mediated via α7 nicotinic acetylcholine receptor (α7nAChR). Suppression of TGFβ1-induced collagen synthesis occurred via an unknown molecular mechanism not involving modulation of the Smad, cAMP, Akt, c-Jun, or MAPK pathway. In vivo, tropisetron not only prevented skin fibrosis but also reduced the collagen content in established dermal fibrosis induced by bleomycin., Conclusion: Tropisetron directly reduces collagen synthesis in HDFs via an α7nAChR-dependent mechanism. The antifibrogenic and antifibrotic effects of this agent observed in a mouse model of bleomycin- induced scleroderma indicate the future potential of tropisetron in the treatment of fibrotic diseases such as scleroderma., (Copyright © 2013 by the American College of Rheumatology.)

The cAMP response element binding protein (CREB) belongs to the CREB/cAMP response element binding modulator/activating transcription factor 1 family of cAMP-dependent transcription factors mediating a regulation of gene transcription in response to cAMP. Chronic stimulation of β-adrenergic receptors and the cAMP-dependent signal transduction pathway by elevated plasma catecholamines play a central role in the pathogenesis of heart failure. Ion channel remodeling, particularly a decreased transient outward current (I(to)), and subsequent action potential (AP) prolongation are hallmarks of the failing heart. Here, we studied the role of CREB for ion channel regulation in mice with a cardiomyocyte-specific knockout of CREB (CREB KO). APs of CREB KO cardiomyocytes were prolonged with increased AP duration at 50 and 70% repolarization and accompanied by a by 51% reduction of I(to) peak amplitude as detected in voltage-clamp measurements. We observed a 29% reduction of Kcnd2/Kv4.2 mRNA in CREB KO cardiomyocytes mice while the other I(to)-related channel subunits Kv4.3 and KChIP2 were not different between groups. Accordingly, Kv4.2 protein was reduced by 37% in CREB KO. However, we were not able to detect a direct regulation of Kv4.2 by CREB. The I(to)-dependent AP prolongation went along with an increase of I(Na) and a decrease of I(Ca,L) associated with an upregulation of Scn8a/Nav1.6 and downregulation of Cacna1c/Cav1.2 mRNA in CREB KO cardiomyocytes. Our results from mice with cardiomyocyte-specific inactivation of CREB definitively indicate that CREB critically regulates the AP shape and duration in the mouse ventricle, which might have an impact on ion channel remodeling in situations of altered cAMP-dependent signaling like heart failure.

Objective: To assess clinical and microbiological outcomes of an Er:YAG laser in comparison with sonic debridement in the treatment of persistent periodontal pockets in a prospective randomized controlled multicentre study design., Material and Methods: A total of 78 patients in supportive periodontal therapy with two residual pockets were included, 58 were available for the whole follow-up period. Root surfaces were instrumented either with a sonic scaler (Sonicflex(®) 2003 L) or with an Er:YAG laser (KEY Laser(®) 3). Clinical attachment levels (CAL), Probing depths (PD), Plaque control record (PCR) and Bleeding on probing (BOP) were assessed at baseline, 13 and 26 weeks after treatment. In addition, microbiological analysis was performed employing a DNA diagnostic test kit (micro-IDent(®) Plus)., Results: Probing depths and CAL were significantly reduced in both groups over time (p < 0.05), without significant differences between the groups (p > 0.05). BOP frequency values decreased significantly within both groups (p < 0.05), with no difference between the laser and the sonic treatment (p > 0.05). PCR frequency values did not change during the observation period (p > 0.05). Microbiological analysis failed to expose any significant difference based on treatment group or period., Conclusion: Employing both sonic and laser treatment procedures during supportive periodontal care, similar clinical and microbiological outcomes can be expected., (© 2012 John Wiley & Sons A/S.)

The cardiac Na(+)/Ca(2+) exchanger (NCX) generates an inward electrical current during SR-Ca(2+) release, thus possibly promoting afterdepolarizations of the action potential (AP). We used transgenic mice 12.5 weeks or younger with cardiomyocyte-directed overexpression of NCX (NCX-Tg) to study the proarrhythmic potential and mechanisms of enhanced NCX activity. NCX-Tg exhibited normal echocardiographic left ventricular function and heart/body weight ratio, while the QT interval was prolonged in surface ECG recordings. Langendorff-perfused NCX-Tg, but not wild-type (WT) hearts, developed ventricular tachycardia. APs and ionic currents were measured in isolated cardiomyocytes. Cell capacitance was unaltered between groups. APs were prolonged in NCX-Tg versus WT myocytes along with voltage-activated K(+) currents (K(v)) not being reduced but even increased in amplitude. During abrupt changes in pacing cycle length, early afterdepolarizations (EADs) were frequently recorded in NCX-Tg but not in WT myocytes. Next to EADs, delayed afterdepolarizations (DAD) triggering spontaneous APs (sAPs) occurred in NCX-Tg but not in WT myocytes. To test whether sAPs were associated with spontaneous Ca(2+) release (sCR), Ca(2+) transients were recorded. Despite the absence of sAPs in WT, sCR was observed in myocytes of both genotypes suggesting a facilitated translation of sCR into DADs in NCX-Tg. Moreover, sCR was more frequent in NCX-Tg as compared to WT. Myocardial protein levels of Ca(2+)-handling proteins were not different between groups except the ryanodine receptor (RyR), which was increased in NCX-Tg versus WT. We conclude that NCX overexpression is proarrhythmic in a non-failing environment even in the absence of reduced K(V). The underlying mechanisms are: (1) occurrence of EADs due to delayed repolarization; (2) facilitated translation from sCR into DADs; (3) proneness to sCR possibly caused by altered Ca(2+) handling and/or increased RyR expression.

Objective: This study was designed to evaluate the clinical outcome of patients undergoing portal vein embolization (PVE) and autologous CD133 bone marrow-derived stem cell (CD133+ BMSC) application before extended right hepatectomy., Background: We have previously shown that portal venous infusion of CD133+ BMSCs substantially increases hepatic proliferation, when compared with PVE alone., Methods: : Among 40 consecutive patients with a median follow-up of 28 months (7.4-57.2) scheduled for extended right hepatectomy, we compared a preconditioned group with PVE and CD133+ BMSC cotreatment (PVE+SC group, n = 11) and a group pretreated only with PVE (PVE group, n = 11). Functional and overall outcomes after extended right hepatectomy were evaluated. Patients without presurgical treatment served as controls (n = 18)., Results: In preconditioned patients, mean hepatic growth of segments II/III 14 days after PVE in the PVE+SC group was significantly higher (138.66 mL ± 66.29) when compared with that of PVE group patients (62.95 mL ± 40.03; P = 0.004). There were no significant differences among all 3 groups regarding general and oncological characteristics and functional parameters on postoperative day (POD) 7. Lack of hepatic preconditioning, extrahepatic extension of resection, and postoperative complications were of negative prognostic value, using univariate analysis (P < 0.05). In multivariate analysis, freedom from postoperative major complications (P = 0.012), coagulation status on POD 7 (international normalized ratio < 1.4; P = 0.027), and presurgical expansion of the future liver remnant volume (P = 0.048) were positively associated with overall survival. Post hoc analysis revealed a better survival for the PVE+SC group (P = 0.028) compared with the PVE group (P = 0.094) and compared with controls., Conclusion: Promising data from this survival analysis suggest that PVE, together with CD133+ BMSC pretreatment, could positively impact overall outcomes after extended right hepatectomy.

The crude extracts of HYPERICUM species native to South Brazil showed analgesic and antidepressant-like effects in rodents. The chemical characterization of these species revealed that they are rich in flavonoids and phloroglucinol derivatives. In the present study a detailed investigation was performed on the activities of hyperoside (HYP), a common flavonoid in the genus HYPERICUM. Hyperoside was obtained from the aerial parts of H. CAPRIFOLIATUM by chromatographic procedures. Mice treated with single doses (10, 20 and 40 mg/kg i.p.) did not present signs of toxicity or weight loss. At 20 and 40 mg/kg i.p. the mice exploratory behavior in the open field test was reduced. At 20 mg/kg i. p. the pentobarbital sleeping time increased, but not the sleeping latency. No activity was found on the hot-plate (10 and 20 mg/kg i.p.) or in the acetic acid-induced writhing test (20 and 40 mg/kg p.o.). Nevertheless, an antidepressant-like effect in the forced swimming test in mice and rats was observed (HYP 10 and 20 mg/kg i.p. in mice; HYP 1.8 mg/kg/day p.o. in rats). The antidepressant-like effect in rats was prevented by the administration of sulpiride (50 mg/kg i.p.) a D2 antagonist. In conclusion, hyperoside was found to present a depressor effect on the central nervous system as well as an antidepressant-like effect in rodents which is, at least in part, mediated by the dopaminergic system., (© Georg Thieme Verlag KG Stuttgart · New York.)

Several species of the genus Hypericum (Guttiferae) have been used for analgesic purposes all over the world and some of them have demonstrated to possess this effect in rodents. This study describes the antinociceptive effect of the cyclohexane extract from aerial parts of H. polyanthemum (POL) as well as of benzopyrans, 6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran (HP1), 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2), and 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3), which are the main components of POL. The antinociceptive effect was evaluated through hot-plate and writhing tests in mice, and the opioid system involvement was assessed by using naloxone (2.5 mg/kg, s.c.) antagonism. In the hot-plate test, POL (45, 90, 180 mg/kg, p.o) showed a dose-dependent effect, and out of the benzopyrans only HP1 (30, 60, 90 mg/kg, i.p.) was active. Its effect was also dose-dependent, with the maximum reached at 60 mg/kg. HP1 60 mg/kg (p.o.) also inhibited acetic acid-induced writhing in 58%. The pretreatment with naloxone abolished the antinociceptive effect of HP1 60 mg/kg (i.p) in the hot plate. Furthermore, the H. polyanthemum cyclohexane extract and HP1 did not affect the mice performance in the rota-rod apparatus suggesting that at antinociceptive doses they do not present gross neurotoxicity nor induce motor impairment. From these data it is reasonable to assume that the benzopyran HP1 accounts for the H. polyanthemum cyclohexane extract antinociceptive effect, and this effect is, at least in part, mediated by an opioid-like mechanism., (© Georg Thieme Verlag KG Stuttgart-New York.)

Purpose: Hepatic resection is established as a safe procedure for colorectal, neuroendocrine, and sarcoma liver metastases. The present study evaluates whether liver resection is an option for patients with non-colorectal, non-neuroendocrine, and non-sarcoma metastases of the liver., Methods: According to data from our prospective clinical tumor registry, we reviewed the medical records of 44 consecutive patients with non-colorectal, non-neuroendocrine, and non-sarcoma liver metastases, who underwent hepatic resection from January 2000 to December 2008. Univariate Kaplan-Meier analysis and a stepwise multivariable Cox regression model were applied., Results: Following hepatic resection, mean overall survival was 21 months, and 5-year survival was 20%. Following hepatic resection, gender, histology, and chemotherapy were of prognostic value in our patient cohort in univariate analysis (p < 0.05). Multivariate survival analysis confirmed chemotherapy (p = 0.002) as an independent prognostic variable. Following initial resection of the primary tumor, synchrone occurrence of metastases, histology, localization of primary, perioperative complications, interval between initial resection of the primary tumor and resection of the metastases, and metastases in follow-up after hepatic resection were of prognostic value in univariate analysis (p < 0.05). Histology (p = 0.017) and interval between resection of the primary and resection of the metastases (p = 0.030) were confirmed as independent prognostic variables in multivariate survival analysis., Conclusions: Hepatic resection seems to be a safe and promising additive for a selective group of patients with non-colorectal, non-neuroendocrine, and non-sarcoma metastases of the liver.

Platelet glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF), and GPVI, which binds collagen, form an adhesion-signaling complex on platelets and mediate platelet adhesion in flowing blood. Platelet activation following engagement of GPIb-IX-V/GPVI by VWF/collagen is critical for initiation and development of a protective thrombus across a site of damaged or exposed endothelium. We examined platelet aggregation and signaling following selective engagement of platelet GPIbalpha (the major ligand-binding subunit of GPIb-IX-V) by a multivalent surface-expressed GPIbalpha-binding VWF-A1 domain on COS-7 cells. COS-7 cells expressing the VWF-A1 domain containing an R543W mutation (a gain-of-function mutation found in Type 2B von Willebrand's Disease) were used as a selective agonist for GPIb-IX-V. When incubated in a cell-to-platelet ratio of up to 1 : 1200, VWF-A1/R543W cells caused rapid, spontaneous aggregation of washed platelets that was GPIbalpha- and alpha(IIb)beta(3)-dependent (blocked by inhibitory anti-VWF-A1, anti-GPIbalpha and anti-alpha(IIb)beta(3) antibodies). Platelet aggregation was also sensitive to inhibitors of Src, phosphoinositide 3-kinase (PI3-kinase) or Syk, confirming a role for these proteins in GPIbalpha-mediated signal transduction. Platelet tyrosine phosphorylation patterns and specific tyrosine phosphorylation of Syk after GPIbalpha engagement by VWF-A1/R543W was comparable to that induced by engagement of GPVI by collagen or collagen-related peptide (CRP). These data indicate signaling events triggered by specific ligation of GPIbalpha can lead to robust platelet activation and help define GPIb-IX-V as both an adhesion and signaling receptor on platelets.

The aerial parts of Hypericum polyanthemum Klotzsch ex Reichardt (Guttiferae) were successively extracted with supercritical carbon dioxide (SC CO(2)) under pressures of 90, 120, 150 and 200 bar at different temperatures (40, 50 and 60 degrees C), and compared with the n-hexane extract obtained by ultrasound-assisted extraction. The samples obtained were examined regarding extraction yield and HPLC quantification of the main secondary metabolites, the benzopyrans HP1 (6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran), HP2 (7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran) and HP3 (5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl) and the phloroglucinol derivative, uliginosin B. SFE presented higher selectivity than the n-hexane maceration, and the best condition to extract the target metabolites has been determined to be at 50 degrees C and for the high molecular-weight compound, uliginosin B, higher pressures were required.

Objective: An alternate approach to the ganglion impar was chosen to minimize the risk of adverse events. Efficacy of the procedure was evaluated., Methods: Charts and computed tomography (CT)-scans of patients who underwent block and neuroablation of the ganglion impar (Walther) between 2003 and 2007 were systematically reviewed with respect to adverse events and efficacy by rating pain intensity. A total of 76 blocks were performed, 48 of them being diagnostic blocks and 28 neuroablations. Chemical destruction was performed with ethanol, if pain recurred despite injection of local anesthetic., Results: Interventional pain therapy was performed in 43 patients (age: 64.6+/-12.4 y, median 49.5 y, range: 36 to 86 y, male/female: 27/16) presenting with perineal pain of unknown origin (n=15), carcinoma of the prostate (n=8), colorectal carcinoma (n=7), postsurgery of thrombosis of perineal veins (n=3), postherpetic neuralgia (n=4), malformation of the spinal cord (n=2), vaginal protrusion (n=2), failed back surgery syndrome (n=1), and ablation of testis (n=1). CT-guided puncture was not associated with any adverse events and resulted in a reduction of numeric rating scale values from 8.2+/-1.6 to 2.2+/-1.6 (P<0.0001, 95% confidence interval 0.5) immediately at discharge and to 2.2+/-1.4 (P<0.0001, 95% confidence interval 0.4) at 4 months on follow up., Discussion: CT-guided block and neuroablation of the ganglion impar (Walther) results in a significant reduction of pain scores and carries virtually no hazards.

Objective: The use of connective tissue grafts is a proven, effective treatment modality. This study examines whether harvesting connective tissue grafts from the palate causes persistent sensory dysfunction., Methods and Materials: Fourteen patients who had received at least 1 graft were included. Neurosensory tests at the palate performed postoperatively included minimal 2-point discrimination, soft-touch discrimination, pin-tactile discrimination, and thermal discrimination. Measurements were taken at 3 sites each: 5, 10, and 15 mm from the palatal tissue margin at canines, premolars, and first molars., Results: Eight patients had 1 graft, and 6 patients had 2 grafts harvested from their palate. Twelve patients had never noticed any dysfunction. Two patients reported a persistent numbness or a rough palatal surface. Statistically significant differences between donor sites and nondonor sites were shown by 2-point discrimination 5 mm from the margin of canines (6.57 mm +/- 3.79 mm at donor sites and 4.71 mm +/- 4.14 mm at nondonor sites, P = .01). Repeated-measures analysis of variance showed statistically significant differences between donor sites and nondonor sites for measurements taken 5 and 10 mm from the margin of all included teeth (P = .02)., Conclusions: Some sensory changes can occur after graft harvesting from the palate.

Background: Cholangiocarcinoma (CC), the most common biliary tract malignancy, is frequently seen in advanced unresectable stages and is typically localized extrahepatically. Early diagnosis is unusual because of nonspecific symptoms. Painless jaundice is usually the first sign of tumor., Method: We present a patient with a CC (Klatskin tumor) with a complete biliary drainage by an aberrant bile duct without jaundice., Results: A 67-year-old woman presented with persisting elevation of liver parameters. Diagnostic tests showed a Klatskin tumor type II. A curative right hepatic trisegmentectomy was performed after liver volume augmentation by preoperative vein embolization., Conclusions: A direct drainage of the right posterior bile duct into the common bile duct as an aberrant hepatic duct is a rare variation and is present in less than 5% of the population. In case of persistently perturbed liver function tests, an aberrant bile duct can cover up severe intrahepatic cholestasis and even obscure the diagnosis of a Klatskin tumor. Up to now it has not been described in the literature.

Portal vein thrombosis is an infrequent entity, which may cause high morbidity and mortality. We report a case of portal vein thrombosis due to benign stenosis following partial pancreatoduodenectomy with segmental replacement of the portal vein by a Gore-tex graft. Using a surgical access to jenunal veins, local thrombolysis, mechanical fragmentation of thrombus, and stent placement were successfully performed.

Neonatal rat cardiomyocytes mainly coexpress the connexins Cx40, Cx43, and to a small amount Cx45, leading to potential formation of mixed (heteromeric/heterotypic) gap junction channels. Using the dual-voltage clamp technique with switching clamp circuits, the authors investigated voltage sensitivity of gap junction channels between cell pairs of Cx40, Cx43, and Cx45 stably transfected HeLa cells and compared those data to data obtained from cell pairs of cultured neonatal rat cardiomyocytes. In accordance to previously published data, the relationship between normalized conductance and transjunctional voltage (g/V(j)) was quasisymmetrical for the transfected HeLa cells, indicating homotypic gap junction channels. Boltzmann curves fitted to data obtained from neonatal rat cardiomyocyte pairs expressing both Cx40 and Cx43 showed an asymmetrical inactivation pattern, which cannot be explained by the presence of pure populations of homotypic gap junction channels of either isoform. In conclusion the authors assume the additional presence of heterotypic and possibly even heteromeric gap junction channels in neonatal rat cardiomyocytes.

We wanted to elucidate whether extracellular calcium may regulate the expression of the cardiac gap-junction proteins connexin 40 and connexin43. In the free wall of the left atria of 126 cardiac surgery patients with either sinus rhythm (SR) or chronic atrial fibrillation (AF), we determined the expression of the cardiac gap-junction proteins Cx43 and Cx40 by Western blot and immunohistology. For deeper investigation, we incubated cultured neonatal rat cardiomyocytes at 2 or 4 mM Ca(++) for 24 h and determined intercellular coupling, Cx40, Cx43 protein and mRNA expression, protein trafficking and sensitivity to verapamil (10-100 nM), cyclosporin A (1 microM),and BMS605401 (100 nM), a specific inhibitor of Ca(2+)-sensing receptor (CaSR). We found in patients that both Cx are up-regulated in AF in the left atrium (by 100-200%). Interestingly, Cx40 was mainly up-regulated, if total serum calcium was >or=2.2 mM, while Cx43 was independent from extracellular [Ca(++)]. In cultured cells, 4 mM Ca(++)-exposure lead to up-regulation of Cx40, but not Cx43. We found enhanced Cx40 in the plasma membrane and reduced Cx40 in the Golgi apparatus. The membrane Cx40 up-regulation resulted in enhanced gap-junction intercellular coupling with a shift in the Boltzmann fit of voltage-dependent inactivation indicating a higher contribution of Cx40 as revealed by dual whole cell voltage clamp experiments. BMS605401 could prevent all Ca(2+)-induced changes. Moreover, cyclosporin A completely abolished the Ca(2+)-induced changes, while verapamil was ineffective. We conclude that extracellular calcium (24 h exposure) seems to up-regulate Cx40 but not Cx43.

Connexin43 (Cx43) is the predominant intercellular gap junction protein in the heart providing intercellular communication for the cell-to-cell transfer of electrical activation. In a previous study, we could show that alpha-adrenoceptor stimulation can affect Cx43 expression and function. We now wanted to elucidate which alpha1-adrenoceptor subtype might be involved. Cultured neonatal rat cardiomyocytes were exposed to various concentrations of phenylephrine (0.1-1,000 nM) for 24 h (n=6). Thereafter, cells were harvested, and after lysis, Cx43 content was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Results were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Finally, we determined the effect of this treatment on intercellular gap junction conductivity using dual whole-cell voltage clamp. Similarly, we tested the effect of an additional treatment with either 10 nM prazosin or, to assess the subtypes, 10 nM of the alpha(1A)-antagonist RS17053 (n=6), 500 nM of the alpha(1B)-antagonist AH1111OA (n=6), or 50 nM of the alpha(1D)-antagonist BMY7378 (n = 6). Moreover, we incubated the cells for 24 h with the alpha(1A)-adrenoceptor agonist A61603 (10 nM). Phenylephrine led to enhanced Cx43 expression with a pEC50 8.00+/-0.06. The other cardiac connexins, Cx40 and Cx45, as well as GAPDH were not affected. This increase in Cx43 expression resulted in enhanced gap-junction conductance (44+/-4 nS vs 26+/-4 nS). As expected, the increased Cx43 expression could be antagonized by prazosin. Moreover, it was nearly completely inhibited by BMY7378 but was not significantly affected by RS17053. AH1111OA led to a moderate but incomplete inhibition. In contrast, beta-actin expression was also up-regulated by phenylephrine but was inhibited by prazosin or RS17053, while it was not affected by BMY7378 or AH1111OA. About 24 h exposure to the alpha(1A)-adrenoceptor agonist A61603 led to a twofold increase in beta-actin but did not affect Cx43. The low pEC50 value of about 1 nM for noradrenaline reported in our earlier study fits well to the hypothesis of an effect mediated predominantly via alpha(1D)-adrenoceptors, which is further supported by the finding of a nearly complete antagonisation of the phenylephrine effect by BMY7378. Thus, we conclude that cardiac Cx43 expression seems to be regulated via alpha(1)-adrenoceptors predominantly by subtype alpha(1D)-adrenoceptors, while other proteins like beta-actin seem to be regulated via alpha(1A)-adrenoceptors.

The leaves of sage (Salvia officinalis L., Lamiaceae) contain high amounts of phenolic diterpenes such as carnosol and carnosic acid. These compounds display antioxidant and anti-inflammatory effects in vitro. Here, we have investigated the anti-inflammatory potency of a sage extract (SE) rich in phenolic diterpenes in vivo using the ultraviolet (UV) erythema test. In a prospective randomised double-blind placebo-controlled study, test areas on the backs of 40 healthy volunteers were irradiated with the 1.5-fold minimal erythema dose. Subsequently, the test areas were treated occlusively with 2% SE in a hydrophilic ointment, compared to 1% hydrocortisone and 0.1% betamethasone as positive controls, and the vehicle alone as placebo. Erythema values were measured photometrically prior to irradiation and after 48 hours. Compared to placebo, SE significantly reduced the ultraviolet-induced erythema, to a similar extent as hydrocortisone. These data suggest that SE might be useful in the topical treatment of inflammatory skin diseases.

Purpose: This randomized clinical trial compared the ability of two dentifrices, one containing 5000 ppm fluoride (Duraphat) and the other 1450 ppm fluoride (Caries Protection, control) to reverse non-cavitated fissure carious lesions., Methods: 30 adults having occlusal lesions with DIAGNOdent values between 10 and 20 were randomly assigned to two groups of 15 participants each. DIAGNOdent measurements were performed after cleaning the teeth with cleaning brushes and after additional cleaning using an airflow device. The participants were instructed to brush their teeth three times a day for 3 minutes after the meals with a 2 cm line of their assigned dentifrice. After a period of 2 weeks, the teeth were cleaned again with the airflow device and laser fluorescence measurement was performed., Results: The group using the dentifrice containing 5000 ppm showed a significantly higher decrease in laser fluorescence (21%) over the test period than the control group (4%; P< 0.001; repeated measures ANOVA). The decrease in laser fluorescence in the test group was significant (P< 0.001; Wilcoxon signed-rank test). The difference between cleaning the teeth with cleaning brushes and additional cleaning with airflow before DIAGNOdent measurement was not statistically different (P> 0.05; Wilcoxon signed-rank test).

This study investigated the efficacy and tolerability of potassium bromide in 113 patients (aged, 1-20 years) with severe epilepsy and generalized tonic-clonic seizures. Potassium bromide was started at 45 mg/kg and raised to 70 mg/kg (median). Steady-state blood level was reached after a median of 28 days (range, 5-95 days). The number of patients who had suffered generalized tonic-clonic seizures during the last month dropped from 82 to 41, and the median frequency, dropped from 4.5 to 0 per month. Of the patients with generalized tonic-clonic seizures during baseline, 49% showed none in the last 4 weeks of the study, and another 31% showed a reduction by more than 50%. Potassium bromide should have a place as a drug of tertiary choice in the treatment of children with epilepsy. Experience with the drug and close clinical and pharmacologic monitoring are necessary to achieve the greatest possible benefit and avoid side effects.