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Adenylyl cyclases (AC) are essential for the normal and pathophysiological response of many cells. In cardiomyocytes, the predominant AC isoforms are AC5 and AC6. Specific AC5 inhibition was suggested as an option for the treatment of heart failure potentially advantageous over β-blockers. We previously reported an interaction between the calcium-binding protein annexin A4 (ANXA4) and AC5 in human embryonic kidney 293 (HEK293) cells and an inhibition of cyclic adenosine monophosphate (cAMP) production in cardiomyocytes. Here, we investigated whether ANXA4 is able to differentiate between AC5 and AC6. In transfected HEK293 cells, ANXA4 specifically co-immunoprecipitated with AC5 and not with AC6, via its N-terminal domain. Both ANXA4 and a peptide comprising the ANXA4 N-terminal sequence (A4N1-22 ) decreased the cAMP production in AC5 and not in AC6 expressing cells. In line with ACs inhibition, in myocytes from ANXA4-deficient mice, β-adrenoceptor (βAR) stimulation led to a higher increase of the L-type calcium current (ICaL ) and to an excessive action potential duration (APD) prolongation as compared to wild-type cardiomyocytes. This enhanced response was reversed in the presence of A4N1-22 peptide likely via specific AC5 inhibition. We conclude that via the N-terminal domain ANXA4 inhibits AC5 not AC6, and that A4N1-22 as a specific AC5 inhibitor could serve as a novel therapeutic tool for the treatment of AC5-linked diseases.

Aims Abnormal intracellular calcium handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signaling pathway contributes to atrial remodeling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models. Methods and results Expression levels of NFATc1-c4 isoforms were assessed by quantitative reverse transcription-polymerase chain reaction in right atrial appendages from patients with chronic AF. NFATc1 and NFATc2 mRNA levels were elevated in chronic AF (cAF) patients compared with those in sinus rhythm (SR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM: NFATc2-/- mice. Programmed electrical stimulation revealed that CREM: NFATc2-/- mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with WT controls, which was reversed in CREM: NFATc2-/- mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM: NFATc2-/- mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on RyR2 in CREM: RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras And EF-Hand Domain-Containing Protein (RASEF) as a direct target of NFATc2 mediated transcription. Conclusion Our findings reveal activation of the NFAT signaling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model. Translational perspective Atrial fibrillation (AF) is a progressive disease characterized by electrical and structural remodeling which promotes atrial arrhythmias. This study provides evidence for increased 'nuclear factor of activated T-cell' (NFAT) signaling in patients with chronic AF. Studies in the CREM transgenic model of progressive AF revealed that the NFATc2 isoform mediates atrial remodeling associated with AF substrate development. Chromatin immunoprecipitation sequencing of atrial biopsies from AF patients identified 'Ras And EF-Hand Domain-Containing Protein' (RASEF) as a downstream target of NFATc2-mediated transcription, suggesting that targeting these factors might be beneficial for curtailing AF progression.

About half of the cardiac serine/threonine phosphatase activity is due to the activity of protein phosphatase type 1 (PP1). The activity of PP1 can be inhibited by an endogenous protein for which the expression inhibitor-2 (I-2) has been coined. We have previously described a transgenic mouse overexpressing a truncated form of I-2. Here, we have described and initially characterized several founders that overexpress the non-truncated (i.e., full length) I-2 in the mouse heart (TG) and compared them with non-transgenic littermates (WT). The founder with the highest overexpression of I-2 displayed under basal conditions no difference in contractile parameters (heart rate, developed tension, and its first derivate) compared to WT. The relative level of PP1 inhibition was similar in mice overexpressing the non-truncated as well as the truncated form of I-2. For comparison, we overexpressed I-2 by an adenoviral system in several cell lines (myocytes from a tumor-derived cell line (H9C2), neonatal rat cardiomyocytes, smooth muscle cells from rat aorta (A7R5)). We noted gene dosage-dependent staining for I-2 protein in infected cells together with reduced PP1 activity. Finally, I-2 expression in neonatal rat cardiomyocytes led to an increase of Ca2+ transients by about 60%. In summary, we achieved immunologically confirmed overexpression of wild-type I-2 in cardiovascular cells which was biochemically able to inhibit PP1 in the whole heart (using I-2 transgenic mice) as well as in isolated cells including cardiomyocytes (using I-2 coding virus) indirectly underscoring the importance of PP1 for cardiovascular function.

Background: Atrial fibrillation (AF) is frequently associated with enhanced inflammatory response. The NLRP3 (NACHT, LRR, and PYD domain containing protein 3) inflammasome mediates caspase-1 activation and interleukin-1β release in immune cells but is not known to play a role in cardiomyocytes (CMs). Here, we assessed the role of CM NLRP3 inflammasome in AF. Methods: NLRP3 inflammasome activation was assessed by immunoblot in atrial whole-tissue lysates and CMs from patients with paroxysmal AF or long-standing persistent (chronic) AF. To determine whether CM-specific activation of NLPR3 is sufficient to promote AF, a CM-specific knockin mouse model expressing constitutively active NLRP3 (CM-KI) was established. In vivo electrophysiology was used to assess atrial arrhythmia vulnerability. To evaluate the mechanism of AF, electric activation pattern, Ca 2+ spark frequency, atrial effective refractory period, and morphology of atria were evaluated in CM-KI mice and wild-type littermates. Results: NLRP3 inflammasome activity was increased in the atrial CMs of patients with paroxysmal AF and chronic AF. CM-KI mice developed spontaneous premature atrial contractions and inducible AF, which was attenuated by a specific NLRP3 inflammasome inhibitor, MCC950. CM-KI mice exhibited ectopic activity, abnormal sarcoplasmic reticulum Ca 2+ release, atrial effective refractory period shortening, and atrial hypertrophy. Adeno-associated virus subtype-9–mediated CM-specific knockdown of Nlrp3 suppressed AF development in CM-KI mice. Finally, genetic inhibition of Nlrp3 prevented AF development in CREM transgenic mice, a well-characterized mouse model of spontaneous AF. Conclusions: Our study establishes a novel pathophysiological role for CM NLRP3 inflammasome signaling, with a mechanistic link to the pathogenesis of AF, and establishes the inhibition of NLRP3 as a potential novel AF therapy approach.

LiNi 1/3 Co 1/3 Mn 1/3 O 2 (NCM) cathode material can be charged to different potentials. When using the standard salt LiPF 6 , elevated cut-off potentials during charge lead to increased capacity and energy density during discharge, but reduce the cycle life dramatically. Metal dissolution is promoted by acids, like HF, derived from deterioration of the electrolyte salt. To exclude the influence of HF, as alternative to LiPF 6 , the less hydrolysis sensitive LiBF 4 and the fluorine-free conducting salt LiClO 4 were investigated. To determine the metal dissolution behavior, NCM electrodes were stored in 1 M EC/DMC (1/1, by wt.) electrolyte containing different salts for 28 days at different states of charge. The measurement of the metal ion content in the electrolyte was carried out by the inductively coupled plasma/optical emission spectrometry (ICP-OES) technique. Constant current cycling experiments were performed with different charge cut-off potentials. In contrast to LiPF 6 , LiBF 4 showed suppressed metal dissolution and superior cycling performance with low capacity fading even at high cut-off potentials (4.6 V vs. Li/Li + ). At lower cut-off potentials, metal dissolution, as well as capacity fading, is rather low for LiClO 4 but escalates at potentials above 4.4 V vs. Li/Li + .

1,3,2-Dioxathiolane-2,2-dioxide (DTD) was investigated as co-solvent for PC based electrolytes in combination with a graphite electrode. An amount of 10wt% of DTD in PC was used to form an effective and stable SEI on graphite during the first cycle. The decomposition of DTD takes place before PC co-intercalation may happen, thus preventing the exfoliation of graphite. Additionally, the electrolyte, 1 M LiBF 4 in PC:DTD (9:1, by wt.) was proven to be compatible with NCM electrodes. Raman spectra confirmed that LiBF 4 completely dissociates into ions in PC:DTD (9:1, by wt.). The electrolyte shows better conductivities in comparison to other LiBF 4 based electrolytes and good rate capability up to 5 C on graphite. Therefore, LiBF 4 can be used as an alternative lithium salt in lithium-ion batteries, in combination with PC based electrolytes.

Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an important cause of sudden cardiac death especially in times of increased sympathetic tone, for example, during sports, which have been confirmed by nuclear imaging studies. However, the underlying biochemical pathways remain to be delineated. Therefore, we investigated the expression levels of proteins of the signaling cascade in patients with ARVC. Methods During diagnostic work-up, right ventricular endomyocardial biopsies (EMBs) were sampled from 15 consecutive male ARVC patients (52 ± 14 years). Tissue levels of key proteins of the signaling cascade were analyzed. Results were compared to those obtained from EMBs of 10 patients with idiopathic right ventricular outflow-tract tachycardia (RVOT; 41 ± 14 years) and of five control subjects without identifiable structural heart disease (42 ± 13 years; P = ns). Results Among the proteins analyzed, only tissue levels of norepinephrine (NE; P < 0.04) and cyclic adenosine-3´,5´-monophospate (cAMP; P < 0.01) were significantly lower in ARVC when compared to RVOT patients. When compared to controls, mean cAMP levels were lower in patients with ARVC but did not reach statistical significance. No differences in cAMP were observed between RVOT and controls. Conclusions The current findings confirm and expand the concept of adrenergic dysfunction in ARVC: the reduction of NE in ARVC could lead to an impaired stimulation of β-adrenoceptor subsequent signaling pathways with potential implication for cardiac fibrosis and arrhythmogenesis.

The composition and formation of the SEI which is formed on lithium, which has been electrochemically deposited on a Cu substrate, is investigated by Raman spectroscopy and mass spectrometry. Semicarbonates, Li2CO3 and Li2C2 are identified as SEI components. The existence of Li2C2 is further proven with mass spectrometry by analyzing the gases which are formed by hydrolysis. Raman mapping reveals that Li2CO3 is homogeneously distributed over the copper substrate, on which lithium was deposited. At contrast, Li2C2 is mainly located on the plated lithium. An in situ Raman spectroscopy method for SEI investigation was developed and shows that Li2C2 is not formed at a potential >0 V vs. Li/Li+.

This work is a comparative study on lithium cycling on copper in solutions based on ethylene carbonate (EC) and diethyl carbonate (DEC) (3:7, by wt.) and two lithium salts, lithium hexafluorophosphate (LiPF 6 ) and lithium difluoro(oxalato)borate (LiDFOB). Coulombic efficiencies of the long term lithium deposition/dissolution experiments and dissolution-rate (D-rate) tests on copper demonstrated clearly the superior behavior of the LiDFOB-based electrolyte. To clarify the impact of the formed solid electrolyte interphase (SEI) on the Coulombic efficiencies achieved in the electrolytes, voltage drop values of the D-rate tests were compared with measured values of conductivities and AC impedance measurements of the electrolytes. The formed SEI has a larger influence on voltage drop values of a cell than the conductivity of the electrolyte. The correlation between surface chemistry, morphology, and Coulombic efficiencies of lithium deposition on copper was investigated by scanning electron microscopy (SEM) and Raman spectroscopy. All methods demonstrated the strong influence of the investigated lithium salts on the lithium deposition/dissolution performance on copper substrates. The LiDFOB-based electrolyte showed superior SEI properties than the LiPF 6 -based electrolyte.

In human atrium, serotonin (5-HT) exerts pleiotropic effects, which are thought to be mediated via 5-HT4 receptors. Here, we used transgenic mice (TG) that overexpress the human 5-HT4(a) receptor under control of the heart-specific α-myosin heavy chain promoter in the atria (and ventricles). Contractile studies were performed in isolated electrically driven left atrial preparations and spontaneously beating right atrial preparation of TG and littermate control mice (wild type (WT)). 5-HT increased force of contraction and phospholamban phosphorylation on serine 16 only in left atrial preparations from TG but not from WT. In contrast, β-adrenoceptor stimulation of left atrial preparations by isoprenaline increased force of contraction with similar pEC50 values and to a similar maximum extent in both TG and WT. The contractile effects of 5-HT in left atrial preparations from TG could be blocked by the 5-HT4 receptor-specific antagonists GR125487 or GR113808. In right atrial preparations from WT and TG, the β-adrenoceptor agonist isoprenaline exerted a positive chronotropic effect with similar pEC50 values and similar maximum effects. Only in right atrial preparations from TG but not WT, 5-HT exerted a positive chronotropic effect that could be attenuated by 5-HT4 receptor-specific antagonists. Finally, in left atrial preparations of TG, a higher incidence of arrhythmias was noted compared to WT. The present data indicate that the human 5-HT4 receptors expressed in mouse atria are functional. This is the first transgenic model to study this human receptor in the atrium ex vivo or in vivo.

Organ-directed gene transfer remains an attractive method for both gaining a better understanding of heart disease and for cardiac therapy. However, virally mediated transfer of gene products into cardiac cells requires prolonged exposure of the myocardium to the viral substrate. Pericardial injection of viral vectors has been proposed and used with some success to achieve myocardial transfection and may be a suitable approach for transfection of atrial myocardium. Indeed, such an organ-specific method would be particularly useful to reverse phenotypes in young and adult genetically altered murine models of cardiac disease. We therefore sought to develop a minimally invasive technique for pericardial injection of substances in mice. Pericardial access in anaesthetised, spontaneously breathing mice was achieved using continuous high-resolution ultrasound guidance. We could demonstrate adequate delivery of injected substances into the murine pericardium. Atrial epicardial and myocardial cells were transfected in approximately one third of mice injected with enhanced green fluorescent protein-expressing adenovirus. Cellular expression rates within individual murine atria were limited to a maximum of 20 %; therefore, expression efficiency needs to be further improved. Minimally invasive, ultrasound-guided injection of viral material appears a technically challenging yet feasible method for selective transfection of atrial epi- and myocardium. This pericardial injection method may be useful in the evaluation of potential genetic interventions aimed at rescuing atrial phenotypes in transgenic mouse models. Electronic supplementary material The online version of this article (doi:10.1007/s00210-012-0815-2) contains supplementary material, which is available to authorized users.

Anti-insect depressant toxins represent a subfamily of scorpion venom-derived β-toxins that are polypeptides composed of 61-65 amino acid residues stabilized by four disulfide bridges. These toxins affect the activation of voltage-sensitive sodium channels (NaScTx) and exhibit the preferential ability to induce flaccid paralysis in insect larvae. Here we demonstrate the recombinant expression of the novel cardiac inotropic peptide (Bj-IP) that was classified as an anti-insect depressant βNaScTx isolated from the venom of Hottentotta judaicus. By using "splicing by overlap extension" (SOE)-PCR, allowing for the first time one step de novo synthesis of long-chain scorpion toxin genes, we generated a codon-optimized DNA fragment of Bj-IP for cloning into the Escherichia coli vector pQE30. Moreover, the gene of interest was fused to a 6xHis coding DNA sequence. Subsequent recombinant expression was performed in E. coli KRX. The purification of the polypeptide was achieved by a combination of NiNTA agarose columns and RP (C(18)) high-performance liquid chromatography. The purified fusion protein was digested with factor Xa resulting in the elution of Bj-IP. The yield of recombinant Bj-IP expression was approximately 4.5 mg per liter of culture. Mass spectrometry confirmed the theoretical total mass of Bj-IP (6608 Da). Tag-free Bj-IP was refolded in guanidine chloride buffer with a glutathione redox system which was supplemented with different additives at 16 °C. Supplementation with 10% glycerol produced Bj-IP folding forms that exhibited reproducible biological activity in mouse cardiomyocytes. Cell contractility was increased by almost 3-fold and decay kinetics were hasten by 47% after administration of Bj-IP. Taken together, here we show the recombinant expression of the functionally active cardiac inotropic peptide Bj-IP, a new βNaScTx from H. judaicus, for promising pharmacological applications. Furthermore, our data suggest that the use of SOE-PCR may help to facilitate in future the high throughput of cloning and/or modification of scorpion toxin genes.

Lithium carbide has been found as possible contamination in high purity lithium metal. Raman spectroscopy clearly shows its presence on the surface as well as in the bulk. Mass spectrometry revealed the formation of acetylene during hydrolization of lithium metal also for the bulk material. The total lithium content of the lithium sample is determined with ICP-OES and the ratio of oxygen to carbon is investigated with EDX for the surface and the bulk. Possible sources of the lithium carbide contamination in battery grade lithium are discussed.

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 ( Ito), 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 Ito 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 Ito-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 Ito-dependent AP prolongation went along with an increase of INa and a decrease of ICa,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.

Methyl tetrafluoro-2-(methoxy) propionate (MTFMP) was evaluated as co-solvent for PC-based electrolytes in combination with graphite electrodes. Already 10 wt% (5.6 mol%) of MTFMP in PC were sufficient to form an effective and stable SEI on graphite as confirmed by battery tests. In the first cycle, the formation of an effective SEI via the decomposition of MTFMP was observed before PC co-intercalation thus preventing the subsequent exfoliation of the graphite. The stability of this SEI was verified by long term cycling tests, which showed that the capacity loss of the graphite-based cell was only 1.3% after 300 cycles at 1 C. The electrolyte, 1 M LiPF 6 in PC:MTFMP (9:1 wt%), showed also a good rate capability up to 5 C on graphite. Therefore, MTFMP can be considered as a new, very promising co-solvent for PC-based electrolytes for use in lithium-ion batteries with graphite anodes.

Very recently, mutations in the TRPM4 gene have been identified in four pedigrees as the cause of an autosomal dominant form of cardiac conduction disease. To determine the role of TRPM4 gene variations, the relative frequency of TRPM4 mutations and associated phenotypes was assessed in a cohort of 160 unrelated patients with various types of inherited cardiac arrhythmic syndro-mes. In eight probands with atrioventricular block or right bundle branch block—five familial cases and three spora-dic cases—a total of six novel and two published TRPM4 mutations were identified. In patients with sinus node dysfunction, Brugada syndrome, or long-QT syndrome, no mutations were found. The novel mutations include six amino acid substitutions and appeared randomly distributed through predicted TRPM4 protein. In addition, eight polymorphic sites including two in-frame deletions were found. Mutations separated from polymorphisms by absence in control individuals and familial cosegregation in some families. In summary, TRPM4 gene mutations appear to play a major role in cardiac conduction disease but not for other related syndromes so far. The phenotypes are variable and clearly suggestive of additional factors modulating the disease phenotype in some patients. Hum Mutat 33:109–117, 2012. © 2011 Wiley Periodicals, Inc.

The high flash point solvent adiponitrile (ADN) was investigated as co-solvent with ethylene carbonate (EC) for use as lithium-ion battery electrolyte. The flash point of this solvent mixture was more than 110 °C higher than that of conventional electrolyte solutions involving volatile linear carbonate components, such as diethyl carbonate (DEC) or dimethyl carbonate (DMC). The electrolyte based on EC:ADN (1:1 wt) with lithium tetrafluoroborate (LiBF 4 ) displayed a conductivity of 2.6 mS cm −1 and no aluminum corrosion. In addition, it showed higher anodic stability on a Pt electrode than the standard electrolyte 1 M lithium hexafluorophosphate (LiPF 6 ) in EC:DEC (3:7 wt). Graphite/Li half cells using this electrolyte showed excellent rate capability up to 5 C and good cycling stability (more than 98% capacity retention after 50 cycles at 1 C ). Additionally, the electrolyte was investigated in NCM/Li half cells. The cells were able to reach a capacity of 104 mAh g −1 at 5 C and capacity retention of more than 97% after 50 cycles. These results show that an electrolyte with a considerably increased flash point with respect to common electrolyte systems comprising linear carbonates, could be realized without any negative effects on the electrochemical performance in Li-half cells.

Background: In patients with Brugada syndrome (BrS), life-threatening ventricular tachyarrhythmias predominantly occur during vagal stimulation at rest or during sleep. Previous imaging studies displayed an impaired autonomic function in BrS patients. However, it remains unclear whether these alterations primarily stem from a reduction of synaptic release of norepinephrine (NE) or an enhanced presynaptic reuptake. Both conditions could lead to reduced NE concentrations in the synaptic cleft. Therefore, we analyzed key components of the sympathoadrenergic signaling pathways in patients with BrS. Methods and Results: Endomyocardial biopsies were obtained from eight BrS patients (seven male; age 49 ± 15 years) and five controls (three male; age 43 ± 13 years; P = ns). The concentrations of NE, epinephrine (Epi), NE transport (NET) carrier protein, cyclic adenosine 5′monophosphate (cyclic adenosine monophosphate [cAMP]), inhibitory G-proteins (Gi1,2α), troponin-I (TNI), and phosphorylated TNI were analyzed. Levels of NET, Gi1,2α, TNI, Epi, and phosphorylated TNI were comparable between the groups. Compared to controls, patients with BrS showed reduced cAMP and NE concentrations. Conclusions: The current findings expand the concept of adrenergic dysfunction in BrS: the reduction of NE in BrS could lead to an impaired stimulation of β-adrenoceptors resulting in a reduction of cAMP and alterations of the subsequent signaling pathway with potential implication for arrhythmogenesis. (PACE 2011; 34:1147–1153)

The salt lithium difluoromono(oxalato)borate (LiDFOB) showed some promising results for lithium-ion-cells. It was synthesized via a new synthetic route that avoids chloride impurities. Here we report the properties of its solutions (solvent blend ethylene carbonate/diethyl carbonate (3:7, mass ratio), including its conductivity, cationic transference number, hydrolysis, Al-current collector corrosion-protection ability and its cycling performance with some electrode materials. Some Al-corrosion studies were also performed with the help of our recently developed computer controlled impedance scanning electrochemical quartz crystal microbalance (EQCM) that proofed to be a useful tool for battery material investigations.

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34+ cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.

Plating-stripping and corrosion tests have been carried out at electrolyte solution containing lithium difluoro(oxalato)borate (LiDFOB) as a lithium salt. For comparison, also electrolytes containing other common borate salts have been investigated. After these tests, the same electrolyte solutions have been used in combination with lithium iron phosphate base electrodes at RT. The results of these studies are reported in this paper.

Background and purpose: Classically, stimulation of muscarinic cholinoceptors exerts negative inotropic and chronotropic effects in the atrium of mammalian hearts. These effects are crucial to the vagal regulation of the heart beat. This effect is assumed to be mediated via GTP binding (G) proteins, because they can be abolished by Pertussis toxin. However, it is unknown which G proteins are involved. Experimental approach: We studied contractility in isolated left or right atrium from genetically manipulated mice with deletion of one of two G proteins, either of the α subunit of Gi2 protein (Gi2α) or of the α subunit of Go protein (Goα). Preparations were stimulated with carbachol alone or after pretreatment with the β-adrenoceptor agonist isoprenaline. For comparison, the effects of carbachol on L-type Ca2+-channels in isolated ventricular cardiomyocytes were studied. Key results: The negative inotropic and chronotropic effects of carbachol alone or in the presence of isoprenaline were identical in atria from knockout or wild-type mice. However, the effect of carbachol on isoprenaline-activated L-type Ca2+-channel in isolated ventricular cardiomyocytes was greatly attenuated in both types of knockout mice studied. Conclusions and implications: These data imply that there is either redundancy of G proteins for signal transduction or that Pertussis toxin-sensitive proteins other than Gi2α and Goα mediate the vagal stimulation in the atrium. Moreover, different G proteins mediate the effect of carbachol in ventricle compared with atrium.

Atrial dilatation is an independent risk factor for thromboembolism in patients with and without atrial fibrillation (AF). In many patients, atrial dilatation goes along with depressed contractile function of the dilated atria. While some mechanisms causing atrial contractile dysfunction in fibrillating atria have been addressed previously, the cellular and molecular mechanisms of atrial contractile remodeling in dilated atria are unknown. This study characterized in vivo atrial contractile function in a goat model of atrial dilatation and compared it to a goat model of AF. Differences in the underlying mechanisms were elucidated by studying contractile function, electrophysiology and sarcoplasmic reticulum (SR) Ca2+ load in atrial muscle bundles and by analyzing expression and phosphorylation levels of key Ca2+-handling proteins, myofilaments and the expression and activity of their upstream regulators. In 7 chronically instrumented, awake goats atrial contractile dysfunction was monitored during 3 weeks of progressive atrial dilatation after AV-node ablation (AV block goats (AVB)). In open chest experiments atrial work index (AWI) and refractoriness were measured (10 goats with AVB, 5 goats with ten days of AF induced by repetitive atrial burst pacing (AF), 10 controls). Isometric force of contraction (FC), transmembrane action potentials (APs) and rapid cooling contractures (RCC, a measure of SR Ca2+ load) were studied in right atrial muscle bundles. Total and phosphorylated Ca2+-handling and myofilament protein levels were quantified by Western blot. In AVB goats, atrial size increased by 18% (from 26.6+/-4.4 to 31.6+/-5.5 mm, n=7 p

Background— Chronic stimulation of the β 1 -adrenoceptor (β 1 AR) plays a crucial role in the pathogenesis of heart failure; however, underlying mechanisms remain to be elucidated. The regulation by transcription factors cAMP response element-binding protein (CREB) and cyclic AMP response element modulator (CREM) represents a fundamental mechanism of cyclic AMP–dependent gene control possibly implicated in β 1 AR-mediated cardiac deterioration. Methods and Results— We studied the role of CREM in β 1 AR-mediated cardiac effects, comparing transgenic mice with heart-directed expression of β 1 AR in the absence and presence of functional CREM. CREM inactivation protected from cardiomyocyte hypertrophy, fibrosis, and left ventricular dysfunction in β 1 AR-overexpressing mice. Transcriptome and proteome analysis revealed a set of predicted CREB/CREM target genes including the cardiac ryanodine receptor, tropomyosin 1α, and cardiac α-actin as altered on the mRNA or protein level along with the improved phenotype in CREM-deficient β 1 AR-transgenic hearts. Conclusions— The results imply the regulation of genes by CREM as an important mechanism of β 1 AR-induced cardiac damage in mice.

Mechanical unloading of failing hearts by left ventricular (LV) assist devices is regularly used as a bridge to transplantation and may lead to symptomatic improvement. The latter has been associated with altered phosphorylation of cardiac regulatory proteins, but the underlying mechanisms remained unknown. Here, we tested whether cardiac unloading alters protein phosphorylation by affecting the corresponding kinase-phosphatase balance. Cardiac unloading and reduction in LV mass were induced by heterotopic heart transplantation in rats for two weeks (n=8). Native in situ hearts from the recipient animals were used as controls (n=8). The steady-state protein kinase A (PKA) and/or Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) phosphorylation levels of phospholamban (PLB, Ser(16) and Thr(17)) and troponin I (TnI, Ser(23/24)) were decreased by 40-60% in unloaded hearts. Consistently, in these hearts PKA activity was decreased by approximately 80% and the activity of protein phosphatase 1 and 2A was increased by 50% and 90%, respectively. In contrast, CaMKII activity was approximately 60% higher, which may serve as a partial compensation. These data indicate that unloading shifts the kinase-phosphatase balance towards net dephosphorylation of PLB and TnI. This shift may also contribute to the reduction in phosphorylation levels of cardiac phosphoproteins observed in diseased human hearts after LVAD.

The actions of adenosine in the human atrium are of clinical relevance, for instance, to stop supraventricular arrhythmias. The purpose of the present study was to re-evaluate the inotropic effect of adenosine in the human atrium. We studied the effect of adenosine (cumulatively applied) on force of contraction in isolated electrically driven right atrial preparations from patients with coronary heart disease undergoing cardiac surgery. It has been known for some time that adenosine via A(1)-adenosine receptors can elicit a negative inotropic effect in human atrial preparations. We report here that in 25% of the patients studied, a positive inotropic effect to adenosine was detectable. The A(1)-adenosine receptor antagonist 1,3-dipropyl-cyclopentyl-xanthine attenuated this effect. Hence, we conclude that at least in some patients, adenosine can lead to a hitherto overlooked positive inotropic effect.

We studied contractile effects in isolated electrically driven (1 Hz) atrial preparations from patients undergoing cardiac bypass surgery. ATP concentration dependently (10, 30, and 100 μM) and rapidly decreased force of contraction (negative inotropic effect, NIE) and thereafter more slowly increased force of contraction. The maximum positive inotropic effect (PIE) at 100 μM ATP amounted to 152% of the predrug value ( n = 9) and was stable and could be washed out fast and completely. The PIE did not affect time parameters of contraction (time to peak tension and time of relaxation). Moreover, a similar NIE and PIE were noted with adenosine 5′- O-(2-thiotriphosphate) (100 μM). In contrast 2-methyl-thio-ATP did not exert a NIE but only a PIE. In a second set of experiments, preparations were first incubated for 30 min with purinoreceptor antagonists and, in their continuous presence, 100 μM ATP was applied. However, the PIE and NIE of ATP could neither be blocked with suramin (100 and 500 μM), pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (50 μM), nor reactive blue 2 (30, 100, and 500 μM), which are known blockers for subtypes of P2 receptors, or 1,3-dipropyl-cyclopentvl-xanthine (1 and 10 μM), a subtype (A1 adenosine) P1 receptor blocker. Likewise, the inhibitor of phospholipase C (PLC) activity (U-73122) and the inhibitor of adenylate cyclase activity (SQ-022563) (10 μM each) failed to affect the NIE and the PIE of ATP. We tentatively suggest that the PIE of ATP might be mediated via P2X4-like receptors. In summary, we describe a novel biphasic effect of ATP on force contraction in the isolated human atrium. It is conceivable that ATP plays a physiological role in the human heart, for instance, after cardiac injury to sustain contractility.

Aim: Several genetically modified mice models were studied so far to investigate the role of cardiac calsequestrin (CSQ2) for the contractile function of the ventricle and for the occurrence of ventricular tachycardia. Using a CSQ2 knockout mouse, we wanted to study also the atrial function of CSQ2.Methods: The influence of CSQ2 on atrial function and, for comparison, ventricular function was studied in isolated cardiac preparations and by echocardiography as well as electrocardiography in mice with deletion of CSQ2.Results: Using deletion of exon 1, we have successfully generated a constitutive knockout mouse of the calsequestrin 2 gene (CSQ2(-/-)). CSQ2 protein was absent in the heart (atrium, ventricle), but also in oesophagus and skeletal muscle of homozygous knockout mice. In 6-month-old CSQ2(-/-) mice, relative left atrial weight was increased, whereas relative heart weight was unchanged. The staircase phenomena in paced left atrial preparations on force of contraction and the post-rest potentiation were different between wild type and CSQ2(-/-) indicative for a decreased sarcoplasmic Ca2+ load and supporting an important role of CSQ2 also in the atrium. The incidence of arrhythmias was increased in CSQ2(-/-). In 2year- old CSQ2(-/-) mice, cardiac hypertrophy and heart failure were noted possibly as a result of chronically increased cytosolic Ca2+ levels.Conclusion: These data suggest a functional role of CSQ2 not only in the ventricle but also in the atrium of mammalian hearts. Loss of CSQ2 function can cause not only arrhythmias, but also cardiac hypertrophy and heart failure.

Nitric oxide (NO) mediates fundamental physiological actions on skeletal muscle. The loss of NO synthase (NOS) from the sarcolemma was assumed to be associated with development of Duchenne muscular dystrophy (DMD). We have, however, recently reported that, in contrast to the commonly accepted view, NOS expression in DMD myofibres is up-regulated. This poses the question of the fibre type-specific NOS expression in DMD muscles and how the NOS expression is related to the regeneration or degeneration status. To address this issue, we examined localization of NOS isoforms I, II and III in skeletal muscles of DMD patients employing immunohistochemical labelling with tyramide signal amplification complemented with enzyme histochemistry. We found that NOS immunolabelling as well as metabolic enzyme activity in DMD muscles were heterogeneously distributed along the fibre length of DMD muscle fibres revealing regenerating and degenerate (hypercontracted) fibres as well as normal segments. Like in normal muscles, positive NOS immunoreactivity was found to be associated with fast-oxidative glycolytic (FOG) phenotype. The regeneration status of NOS-positive segments was deduced from the presence of neonatal and developmental myosin heavy chains. High NOS expression in regenerating DMD muscle fibres can be well reconciled with reports about the protective role of endogenous NO in inflammatory diseases and in muscle repair.

Reduced function of the cardiac ryanodine receptor or calsequestrin causes catecholaminergic ventricular tachycardia (VT). These proteins regulate sarcoplasmic Ca(2+) release in close conjunction with two accessory proteins, triadin and junctin. Based on data from cardiomyocytes, we hypothesized that enhanced triadin expression could cause VT. We assessed arrhythmias and electrophysiological changes in vivo and in the beating heart in mice expressing junctin, triadin, or both proteins (TRDxJCN), and measured calcium transients in isolated ventricular cardiomyocytes. TRDxJCN mice were studied to compensate the down-regulation of junctin expression in triadin-expressing mice. Exercise or stress provoked repetitive VT in freely roaming TRDxJCN mice whenever heart rate increased above approximately 600 bpm (p0.05 vs. the three other genotypes). TRDxJCN mice expressed total triadin 2.9-fold (p0.05) and total junctin not different to wildtype (p=ns). Left ventricular systolic function was not different between lineages. beta-adrenoreceptor stimulation (orciprenaline 1.7 microM) provoked early-coupled ventricular ectopy and repetitive VT in isolated, Langendorff-perfused TRDxJCN hearts (p0.05). Under conditions associated with VT (high pacing rate, catecholamine stimulation), action potential duration was shorter in TRDxJCN with VT than in the other genotypes and shorter than in TRDxJCN hearts without VT (p0.05). Ca(2+) transient duration was prolonged in Indo1-loaded TRDxJCN cardiomyocytes under VT-provoking conditions. Action potential prolongation by mexiletine (2 microM or 4 microM) or clarithromycine (150 microM) suppressed VT. Expression of triadin provokes stress- and tachycardia-related ventricular arrhythmias in mice. An imbalance between prolonged intracellular calcium release and shortening of the ventricular action potential may contribute to genesis of arrhythmias in this model.

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 Ca2+ releases was increased in TG accompanied by an enhanced transduction rate of sub-threshold Ca2+ waves into these supra-threshold events. As a likely cause we discovered enhanced NCX-mediated Ca2+ transport and NCX1 protein level in TG. An increase in INCX and decrease in Ito 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. Electronic supplementary material The online version of this article (doi:10.1007/s00395-016-0532-y) contains supplementary material, which is available to authorized users.

Transgenic mice are often used to study the physiologic role of a known gene. The design of experiments with transgenic mice usually does not take into account strain and sex differences, at least in isolated vessels. Therefore, we have compared the contractile response of isolated aortae and isolated pulmonary arteries of male and female mice of different strains (CD1, BL6, and DBA). Contractile stimulation was achieved by depolarization due to KCl, alpha1-adrenoceptor stimulation by phenylephrine and inhibition of protein phosphatase activity by cantharidin. In isolated aorta, strain-specific differences in contractility and sex-specific differences could be observed. The concentration of phenylephrine (PE) inducing half maximal contraction (EC50) was different between aortae from DBA male mice and the other strains tested. Phasic contractions of isolated aortic rings due to PE were seen in all mice except DBA male. In isolated pulmonary arteries, strain-specific differences and sex-specific differences could be observed. The EC50-values of PE were not different between all groups. Phasic contractions due to PE were only seen in pulmonary arteries from CD1 male and BL6 female. In conclusion, strain- and sex-specific differences should be considered in selecting mice used for transgenesis or gene targeting experiments.

In cardiac muscle, junctin forms a quaternary protein complex with the ryanodine receptor (Ry R), calsequestrin, and triadin 1 at the luminal face of the junctional sarcoplasmic reticulum (j SR). By binding directly the Ry R and calsequestrin, junctin may mediate the Ca2+-dependent regulatory interactions between both proteins. To gain more insight into the underlying mechanisms of impaired contractile relaxation in transgenic mice with cardiac-specific overexpression of junctin (TG), we studied cellular Ca2+ handling in these mice. We found that the SR Ca2+ load was reduced by 22 percent in cardiomyocytes from TG mice. Consistent with this, the frequency of Ca2+ sparks was diminished by 32 percent. The decay of spontaneous Ca2+ sparks was prolonged by 117 percent in TG. This finding was associated with a lower Na+-Ca2+ exchanger (NCX) protein expression (by 67 percent) and a higher basal Ry R phosphorylation at Ser2809 (by 64 percent) in TG. The shortening- and Δ(Ca)i-frequency relationships (0.5–4 Hz) were flat in TG compared to wild-type (WT) which exhibited a positive staircase for both parameters. Furthermore, increasing stimulation frequencies hastened the time of relaxation and the decay of (Ca)i by a higher percentage in TG. We conclude that the impaired relaxation in TG may result from a reduced NCX expression and/or a higher SR Ca2+ leak. The altered shortening-frequency relationship in TG seems to be a consequence of an impaired excitation–contraction coupling with depressed SR Ca2+release at higher rates of stimulation. Our data suggest that the more prominent frequency-dependent hastening of relaxation in TG results from a stimulation of SR Ca2+ transport reflected by corresponding changes of (Ca)i.

Skeletal muscle functions regulated by NO are now firmly established. However, the literature on the compartmentalization of NO signaling in myocytes is highly controversial. To address this issue, we examined localization of enzymes engaged in L-arginine-NO-cGMP signaling in the rat quadriceps muscle. Employing immunocytochemical labeling complemented with tyramide signal amplification and electron microscopy, we found NO synthase expressed not only in the sarcolemma, but also along contractile fibers, in the sarcoplasmic reticulum and mitochondria. The expression pattern of NO synthase in myocytes showed striking parallels with the enzymes engaged in L-arginine-NO-cGMP signaling (arginase, phosphodiesterase, and soluble guanylyl cyclase). Our findings are indicative of an autocrine fashion of NO signaling in skeletal muscles at both cellular and subcellular levels, and challenge the notion that the NO generation is restricted to the sarcolemma.

Background— Although downregulation of L-type Ca 2+ current ( I Ca,L ) in chronic atrial fibrillation (AF) is an important determinant of electrical remodeling, the molecular mechanisms are not fully understood. Here, we tested whether reduced I Ca,L in AF is associated with alterations in phosphorylation-dependent channel regulation. Methods and Results— We used whole-cell voltage-clamp technique and biochemical assays to study regulation and expression of I Ca,L in myocytes and atrial tissue from 148 patients with sinus rhythm (SR) and chronic AF. Basal I Ca,L at +10 mV was smaller in AF than in SR (−3.8±0.3 pA/pF, n=138/37 [myocytes/patients] and −7.6±0.4 pA/pF, n=276/86, respectively; P 1c and regulatory β 2a channel subunits were not different. In both groups, norepinephrine (0.01 to 10 μmol/L) increased I Ca,L with a similar maximum effect and comparable potency. Selective blockers of kinases revealed that basal I Ca,L was enhanced by Ca 2+ /calmodulin-dependent protein kinase II in SR but not in AF. Norepinephrine-activated I Ca,L was larger with protein kinase C block in SR only, suggesting decreased channel phosphorylation in AF. The type 1 and type 2A phosphatase inhibitor okadaic acid increased basal I Ca,L more effectively in AF than in SR, which was compatible with increased type 2A phosphatase but not type 1 phosphatase protein expression and higher phosphatase activity in AF. Conclusions— In AF, increased protein phosphatase activity contributes to impaired basal I Ca,L . We propose that protein phosphatases may be potential therapeutic targets for AF treatment.

The influence of pertussis toxin on the effects of adenosine, the adenosine receptor agonist (-)-N6-phenylisopropyladenosine (PIA) and the m-cholinoceptor agonist carbachol on heart rate and atrioventricular (AV) conduction was investigated in spontaneously beating isolated perfused guinea-pig hearts. In addition, the effects of the agents on the electrocardiogram recorded from anesthetized guinea pigs were studied. Adenosine (0.1-100 mumol/l) and PIA (0.001-100 mumol/l) had concentration-dependent negative chronotropic and negative dromotropic effects. These effects were prevented by pretreatment of the animals with pertussis toxin (150 micrograms/kg; i.v.). Carbachol (0.001-100 mumol/l) had similar cardiac depressant effects. These effects were also abolished by pertussis toxin. In contrast, the negative chronotropic and negative dromotropic effects of the calcium antagonist verapamil which was investigated for comparison were not influenced by pretreatment with pertussis toxin. Since the cardiac depressant effects mediated via adenosine receptors or via m-cholinoceptors are most probably due to an activation of a K+ conductance, it is concluded that both receptors in the sinus node and in the AV node may be coupled via a common pertussis toxin-sensitive guanine nucleotide-binding protein to the K+ channel. It remains to be elucidated whether an additional inhibitory coupling to Ca2+ channels also plays a role.

Objective: The aim of the present study was to assess the effects of A1-adenosine receptor (A1-AR) stimulation in ventricle of A1-adenosine receptor overexpressing mice (transgenic mice, TG). Methods: Effects of the A1-adenosine receptor agonist R-PIA ((−)- N 6-phenylisopropyladenosine) on phosphorylation of phospholamban (PLB), Ca2+ transients, Ca2+ currents and cell shortening were studied in isolated ventricular cardiomyocytes. Results: R-PIA alone did not affect contractility in isolated electrically stimulated cardiomyocytes from wild-type mice (WT) or TG. However, after pre-stimulation of β-adrenoceptors by isoproterenol, R-PIA reduced contractility in cardiomyocytes from WT but increased contractility in TG. Under the same conditions, R-PIA reduced isoproterenol-stimulated currents through L-type Ca2+ channels, Ca2+ transients and phosphorylation of PLB in cardiomyocytes from WT. In contrast, R-PIA diminished phospholamban phosphorylation induced by isoproterenol but augmented isoproterenol-elevated currents through L-type Ca2+ channels, and isoproterenol-heightened Ca2+ transients in cardiomyocytes from TG. Conclusions: We suggest that A1-adenosine receptor overexpression reverses the interaction of β-adrenergic and A1-adenosine receptor stimulation, at least in part. Hence, the receptor/effector coupling is dependent on receptor density in this model.

In general, the failing human heart is characterized by a selective reduction in beta(1)-adrenoceptors (beta(1)-ARs) without change in beta(2)-AR density. Medical imaging techniques, either single photon emission computed tomography (SPECT) or positron emission tomography (PET) with appropriate radioligands, offer the possibility of assessing beta-adrenoceptor density non-invasively in humans. To date, neither a SPECT nor a PET radioligand is available for the selective imaging of cardiac beta(1)-ARs. The aim of this study was to develop potential high affinity beta(1)-selective AR radioligands for the non-invasive in vivo imaging of the beta(1)-AR density in the human heart using SPECT or PET. A variety of racemic N-aryl-N'-[2-[3-aryloxy-2-hydroxy-propylamino]-ethyl]-urea derivatives and chain-elongated analogues, related to the established beta(1)-AR antagonist, ICI 89,406 8i, were synthesized. Competition studies using the non-selective AR ligand, [(125)I]iodocyanopindolol ([(125)I]ICYP), and ventricular membrane preparations of wild-type mice revealed nine ligands with higher beta(1)-AR affinities (up to 76-fold) and beta(1)-AR selectivities (up to 139-fold) than 8i. Mostly, these ligands possess a 2-substituted phenoxy group and a 4-substituted phenyl residue in contrast to the lead compound 8i. The non-radioactive counterparts of the desired SPECT- and PET-radiotracers were synthesized as reference compounds [e.g., 8f, 8g, 8h and 8l as the non-radioactive analogues of the radioiodinated SPECT radioligands, 8e and 8h as the non-radioactive compounds of C-11 labelled PET-tracers (C-11 in the methoxy group)]. The established library of high affinity beta(1)-selective AR antagonists was screened for chemical precursors for the radiosynthesis of the mentioned radioligands. Furthermore, the library consists of some comparison compounds that are unsubstituted, allyl- and alkyl-substituted or chain-elongated (e.g., 8a, 8j, 8o and 8r-t). Future steps will include radiolabelling and pharmacokinetic evaluation of the beta(1)-selective target compounds, which could be applied as sympathetic innervation agents for in vivo investigations and diagnostics in patients suffering from cardiac diseases like heart failure and ventricular arrhythmias.

c ¨ Medizinische Klinik B und Zentrale Projektgruppe Ultrastrukturforschung am Gerhard -Domagk-Institut f ur Pathologie, ¨¨ ¨ Westf alische Wilhelms-Universitat, 48149 Munster, Germany d ¨¨ ¨ Medizinische Klinik und Poliklinik C , Westf alische Wilhelms-Universitat, 48149 Munster, Germany e ¨¨ ¨ ¨ Klinik und Poliklinik f ur Nuklearmedizin , Westf alische Wilhelms-Universitat, 48149 Munster, Germany f Abstract Objective: Junctin is a major transmembrane protein in cardiac junctional sarcoplasmic reticulum, which forms a quaternary complex 21 with the ryanodine receptor (Ca release channel), triadin, and calsequestrin. Methods: To better understand the role of junctin in excitation-contraction coupling in the heart, we generated transgenic mice with targeted overexpression of junctin to mouse heart, using the a-MHC promoter to drive protein expression. Results: The protein was overexpressed 10-fold in mouse ventricles and overexpression was accompanied by cardiac hypertrophy (19%). The levels of two other junctional SR-proteins, the ryanodine receptor and triadin, were 3 reduced by 32% and 23%, respectively. However, ( H)ryanodine binding and the expression levels of calsequestrin, phospholamban and 21 SERCA2a remained unchanged. Cardiomyocytes from junctin-overexpressing mice exhibited impaired relaxation: Ca transients decayed at a slower rate and cell relengthening was prolonged. Isolated electrically stimulated papillary muscles from junctin- overexpressing hearts exhibited prolonged mechanical relaxation, and echocardiographic parameters of relaxation were prolonged in the 21 living transgenic mice. The amplitude of caffeine-induced Ca transients was lower in cardiomyocytes from junctin-overexpressing 21 21 21 mice. The inactivation kinetics of L-type Ca channel were prolonged in junctin-overexpressing cardiomyocytes using Ca or Ba as charge carriers. Conclusion: Our data provide evidence that cardiac-specific overexpression of junctin is accompanied by impaired 21

To investigate whether altered function of adenosine receptors could contribute to sinus node or atrioventricular (AV) nodal dysfunction in conscious mammals, we studied transgenic (TG) mice with cardiac-specific overexpression of the A1 adenosine receptor (A1AR). A Holter ECG was recorded in seven freely moving littermate pairs of mice during normal activity, exercise (5 min of swimming), and 1 h after exercise. TG mice had lower maximal heart rates (HR) than wild-type (WT) mice (normal activity: 437 ± 18 vs. 522 ± 24 beats/min, P < 0.05; exercise: 650 ± 13 vs. 765 ± 28 beats/min, P < 0.05; 1 h after exercise: 588 ± 18 vs. 720 ± 12 beats/min, P < 0.05; all values are means ± SE). Mean HR was lower during exercise (589 ± 16 vs. 698 ± 34 beats/min, P < 0.05) and after exercise (495 ± 16 vs. 592 ± 27 beats/min, P < 0.05). Minimal HR was not different between genotypes. HR variability (SD of RR intervals) was reduced by 30% ( P < 0.05) in TG compared with WT mice. Pertussis toxin ( n = 4 pairs, 150 μg/kg ip) reversed bradycardia after 48 h. TG mice showed first-degree AV nodal block (PQ interval: 42 ± 2 vs. 37 ± 2 ms, P < 0.05), which was diminished but not abolished by pertussis toxin. Isolated Langendorff-perfused TG hearts developed spontaneous atrial arrhythmias (3 of 6 TG mice vs. 0 of 9 WT mice, P < 0.05). In conclusion, A1AR regulate sinus nodal and AV nodal function in the mammalian heart in vivo. Enhanced expression of A1AR causes sinus nodal and AV nodal dysfunction and supraventricular arrhythmias.

It was investigated how A1-adenosine receptor overexpression alters the effects of carbachol on force of contraction and beating rate in isolated murine atria. Moreover, the influence of pertussis toxin on the inotropic and chronotropic effects of adenosine and carbachol in A1-adenosine receptor overexpressing atria was studied. Adenosine and carbachol alone exerted negative inotropic and chronotropic effects in electrically driven left atrium or spontaneously beating right atrium of wild-type mice. These effects were abolished or reversed by pre-treatment of animals with pertussis toxin which can interfere with signal transduction through G-proteins. Adenosine and carbachol exerted positive inotropic but negative chronotropic effects in atrium overexpressing A1-adenosine receptors from transgenic mice. The positive inotropic effects of adenosine and carbachol were qualitatively unaltered whereas the negative chronotropic effects were abolished or reversed in atrium overexpressing A1-adenosine receptors after pre-treatment by pertussis toxin. Qualitatively similar effects for adenosine and carbachol were noted in the presence of isoprenaline, β-adrenoceptor agonist. It is concluded that overexpression of A1-adenosine receptors also affects the signal transduction of other heptahelical, G-protein coupled receptors like the M-cholinoceptor in the heart. The chronotropic but not the inotropic effects of adenosine and carbachol in transgenic atrium were mediated via pertussis toxin sensitive G-proteins. Keywords: A1-adenosine receptors, chronotropy, inotropy, M-cholinoceptors, transgenic mice Introduction Adenosine (ado) is generated in the heart in response to stimuli such as hypoxia and β-adrenergic stimulation (Bardenheuer et al., 1987; Berne, 1983). Adenosine exerts negative inotropic and chronotropic effects in the heart (Ralevic & Burnstock, 1998; Schrader et al., 1977; Shryock & Belardinelli, 1997), mediated through the A1-adenosine receptor. In addition to A1-adenosine receptors, A2a-, A2b- and A3-adenosine receptors have been detected in the heart (Boknik et al., 1997; Reppert et al., 1991; Salvatore et al., 1993). In the atrium of most mammals (guinea-pig, mouse, man, rat) ado reduces force of contraction and the beating rate when given alone (Bohm et al., 1984; 1985; 1989; Dobson, 1983; Drury & Szent-Gyorgyi, 1929). Adenosine in the presence of isoprenaline exerts a negative inotropic and chronotropic effect in the atrium of many mammalian species (Ralevic & Burnstock, 1998). The negative inotropic effect in atrium (and in the ventricle after β-adrenergic stimulation) may involve inhibition of adenylyl cyclase activity, inhibition of cAMP-dependent protein kinase, activation of protein phosphatases, stimulation of phospholipase C or opening of ion channels (for review: Stein et al., 1998). More specifically, the negative inotropic effect of ado in atrium may be due to activation of voltage dependent KAch-channels, subsequent shortening of action potential duration and therefore less time for influx of Ca2+ (Belardinelli & Isenberg, 1983). Thus, less Ca2+ is in the cell, and therefore less force can be generated by Ca2+ acting on myofilaments. Transgenic mice have been engineered that overexpress the A1-adenosine receptor (A1-AR) selectively in the heart (Matherne et al., 1997). The transgenic mice were significantly more resistant to the functional and metabolic effects of ischaemia through their overexpressed A1-AR (Matherne et al., 1997; Headrick et al., 1998). In addition, the transgenic overexpression of A1-AR mimicked ischaemic preconditioning in hearts of transgenic animals (Morrison et al., 2000). This cardioprotection is mediated by ATP-sensitive potassium channels (Headrick et al., 2000). In our previous work we demonstrated that ado can reduce the contractility in isolated left atrial preparations from wild-type mice. Moreover, ado exerted a negative chronotropic effect in right atrial preparations from wild-type mice. In mice that overexpress the A1-AR (in this work called transgenic mice) ado still reduced the beating rate in right atrial preparations. However, ado raised force of contraction (positive inotropic effect) in left atrial preparations from transgenic mice (Neumann et al., 1999). It is conceivable that the reversal of the negative inotropic effect of ado to a positive inotropic effect could reside at the level of ado receptors or in alterations in the post-receptor transduction pathway that is common to the A1-AR and the muscarinic cholinoceptor. Therefore carbachol was studied in parallel to ado in this work. The direct and indirect effects of ado and carbachol (carb) in the heart are mediated by inhibitory Gi-proteins, which can be covalently modified and blocked by pertussis toxin (Bohm et al., 1986; 1988; Kurachi et al., 1986; Neumann et al., 1994). The purpose of this study was to investigate whether A1-AR overexpression influences the inotropic and chronotropic effects of M-cholinoceptor stimulation (by carb) in atrium. Furthermore, we wanted to know whether the inotropic and chronotropic effects of ado and carb in atrium are sensitive to pertussis toxin (ptx) pre-treatment of animals that overexpress A1-AR.

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-Forster 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.

Electrolyte solutions, containing the lithium sulfonyl methide salts lithium-tris(trifluoromethanesulfonyl)methide (LiTFSM) and lithium-[bis(trifluoromethylsulfonyl)-pentafluoroethylsulfonyl]methide (LiPFSM) dissolved in organic carbonate solvents, were electrochemically investigated in Li/graphite, Li/LiNi1/3Co1/3Mn1/3O2 (NCM) half-cells and compared to the LiPF6 based electrolyte with regard to their ionic conductivity, electrochemical stability, thermal stability at 60°C and the anodic dissolution behavior vs. Al. While the investigated salts show almost the same performance in Li/graphite half-cells compared to the LiPF6 containing electrolyte, the methide salts show very promising results in Li/NCM half-cells, which depict superior capacity retention as well as higher Coulombic efficiencies compared to the LiPF6 containing electrolyte. Taking the limited anodic stability as well as the occurring anodic dissolution into account, both salts but especially LiTFSM indicate the applicability in lithium ion battery electrolytes for cells with a cutoff potential up to 4.3 V vs. Li/Li+.