Jan S. Schulte

Overexpression of cAMP-response element modulator causes abnormal growth and development of the atrial myocardium resulting in a substrate for sustained atrial fibrillation in mice

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.

Universal cardiac induction of human pluripotent stem cells in two and three-dimensional formats: implications for in vitro maturation.

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. Stem Cells 2015;33:1456–1469

Cardiac Function Is Regulated by B56α-mediated Targeting of Protein Phosphatase 2A (PP2A) to Contractile Relevant Substrates

Background: PP2A is a regulator of cardiac excitation-contraction coupling. Results: Cardiomyocyte-directed overexpression of B56α, the main cardiac PP2A regulatory subunit, results in the dephosphorylation of myofilament proteins, increased Ca2+ sensitivity, and higher contractility. Conclusion: This suggests an important role for B56α in regulating PP2A activity and thereby the contractile function. Significance: PP2A-B56α is a potential pharmacological target to improve cardiac performance in failing hearts. 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 Ser16 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 Ca2+ sensitivity and increased basal contractility in TG hearts. These effects were reversed by β-adrenergic stimulation.

Tropisetron suppresses collagen synthesis in skin fibroblasts via α7 nicotinic acetylcholine receptor and attenuates fibrosis in a scleroderma mouse model

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.

CREB critically regulates action potential shape and duration in the adult mouse ventricle

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.

Suppression of Early and Late Afterdepolarizations by Heterozygous Knockout of the Na+/Ca2+ Exchanger in a Murine Model.

Background—The Na+/Ca2+ 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 Ca2+ imaging techniques. Action potential duration was shorter in hetKO with IKtot not being increased. The rate of spontaneous Ca2+ 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 Ca2+ 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 Ca2+-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.

Sphingosine‐1‐Phosphate Receptor 1 Regulates Cardiac Function by Modulating Ca2+ Sensitivity and Na+/H+ Exchange and Mediates Protection by Ischemic Preconditioning

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 Ca2+ 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 Ca2+ 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 Ca2+ homeostasis by maintaining basal NHE‐1 activity and controls simultaneously myofibril Ca2+ 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.

Universal Cardiac Induction of Human Pluripotent Stem Cells in 2D and 3D formats - Implications for In-Vitro Maturation

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. Stem Cells 2015;33:1456–1469

A mobile data collection framework for the Sensor Web

In the idea of the Sensor Web, sensors should be discoverable, accessible and taskable over the internet. In this paper, we present a mobile data collection framework for connecting wireless sensors with the Sensor Web by using mobile devices. The proposed system is an extension to Locawe platform including now mapping and WSN features together with features of SWE especially Sensor Observation Service (SOS) specified by the Open Geospatial Consortium. Therefore, the Locawe platform running on mobile devices is used to connect to 6LoWPAN sensors, store the measurements of the sensors on the devices memory, convert it into sensor web protocols and insert the measurements into a sensor service as soon as wireless internet connection is available. As a proof of concept, the system is used within an air quality monitoring scenario.

Cardiac expression of the CREM repressor isoform CREM-IbΔC-X in mice leads to arrhythmogenic alterations in ventricular cardiomyocytes

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.