Atrial proteomic profiling reveals a switch towards profibrotic gene expression program in CREM-IbΔC-X mice with persistent atrial fibrillation.

BACKGROUND: Overexpression of the CREM (cAMP response element-binding modulator) isoform CREM-IbΔC-X in transgenic mice (CREM-Tg) causes the age-dependent development of spontaneous AF. PURPOSE: To identify key proteome signatures and biological processes accompanying the development of persistent AF through integrated proteomics and bioinformatics analysis. METHODS: Atrial tissue samples from three CREM-Tg mice and three wild-type littermates were subjected to unbiased mass spectrometry-based quantitative proteomics, differential expression and pathway enrichment analysis, and protein-protein interaction (PPI) network analysis. RESULTS: A total of 98 differentially expressed proteins were identified. Gene ontology analysis revealed enrichment for biological processes regulating actin cytoskeleton organization and extracellular matrix (ECM) dynamics. Changes in ITGAV, FBLN5, and LCP1 were identified as being relevant to atrial fibrosis and structural based on expression changes, co-expression patterns, and PPI network analysis. Comparative analysis with previously published datasets revealed a shift in protein expression patterns from ion-channel and metabolic regulators in young CREM-Tg mice to profibrotic remodeling factors in older CREM-Tg mice. Furthermore, older CREM-Tg mice exhibited protein expression patterns reminiscent of those seen in humans with persistent AF. CONCLUSIONS: This study uncovered distinct temporal changes in atrial protein expression patterns with age in CREM-Tg mice consistent with the progressive evolution of AF. Future studies into the role of the key differentially abundant proteins identified in this study in AF progression may open new therapeutic avenues to control atrial fibrosis and substrate development in AF.

Atrial Proteomic Profiling Reveals a Switch Towards Profibrotic Gene Expression Program in CREM-IbΔC-X Mice with Persistent Atrial Fibrillation.

Background: Overexpression of the CREM (cAMP response element-binding modulator) isoform CREM-IbΔC-X in transgenic mice (CREM-Tg) causes the age-dependent development of spontaneous AF. Purpose: To identify key proteome signatures and biological processes accompanying the development of persistent AF through integrated proteomics and bioinformatics analysis. Methods: Atrial tissue samples from three CREM-Tg mice and three wild-type littermates were subjected to unbiased mass spectrometry-based quantitative proteomics, differential expression and pathway enrichment analysis, and protein-protein interaction (PPI) network analysis. Results: A total of 98 differentially expressed proteins were identified. Gene ontology analysis revealed enrichment for biological processes regulating actin cytoskeleton organization and extracellular matrix (ECM) dynamics. Changes in ITGAV, FBLN5, and LCP1 were identified as being relevant to atrial fibrosis and remodeling based on expression changes, co-expression patterns, and PPI network analysis. Comparative analysis with previously published datasets revealed a shift in protein expression patterns from ion-channel and metabolic regulators in young CREM-Tg mice to profibrotic remodeling factors in older CREM-Tg mice. Furthermore, older CREM-Tg mice exhibited protein expression patterns that resembled those of humans with persistent AF. Conclusions: This study uncovered distinct temporal changes in atrial protein expression patterns with age in CREM-Tg mice consistent with the progressive evolution of AF. Future studies into the role of the key differentially abundant proteins identified in this study in AF progression may open new therapeutic avenues to control atrial fibrosis and substrate development in AF.

Pathophysiological Mechanisms of Cardiac Dysfunction in Transgenic Mice with Viral Myocarditis.

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.

Genetic inhibition of nuclear factor of activated T-cell c2 prevents atrial fibrillation in CREM transgenic mice.

AIMS: Abnormal intracellular calcium (Ca2+) 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) signalling pathway contributes to atrial remodelling. 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 (cAF). NFATc1 and NFATc2 mRNA levels were elevated in cAF patients compared with those in normal sinus rhythm (NSR). 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 wild-type 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 ryanodine receptor type 2 (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 signalling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model.

The first versatile human iPSC-based model of ectopic virus induction allows new insights in RNA-virus disease.

A detailed description of pathophysiological effects that viruses exert on their host is still challenging. For the first time, we report a highly controllable viral expression model based on an iPS-cell line from a healthy human donor. The established viral model system enables a dose-dependent and highly localized RNA-virus expression in a fully controllable environment, giving rise for new applications for the scientific community.

Evidence for Arrhythmogenic Effects of A2A-Adenosine Receptors.

Adenosine can be released from the heart and may stimulate four different cardiac adenosine receptors. A receptor subtype that couples to the generation of cyclic adenosine monophosphate (cAMP) is the A2A-adenosine receptor (A2A-AR). To better understand its role in cardiac function, we studied mechanical and electrophysiological effects in transgenic mice that overexpress the human A2A-AR in cardiomyocytes (A2A-TG). We used isolated preparations from the left atrium, the right atrium, isolated perfused hearts with surface electrocardiogram (ECG) recording, and surface body ECG recordings of living mice. The hypothesized arrhythmogenic effects of transgenicity per se and A2A-AR stimulation were studied. We noted an increase in the incidence of supraventricular and ventricular arrhythmias under these conditions in A2A-TG. Moreover, we noted that the A2A-AR agonist CGS 21680 exerted positive inotropic effect in isolated human electrically driven (1 Hz) right atrial trabeculae carneae. We conclude that A2A-ARs are functional not only in A2A-TG but also in isolated human atrial preparations. A2A-ARs in A2A-TG per se and their stimulation can lead to cardiac arrhythmias not only in isolated cardiac preparations from A2A-TG but also in living A2A-TG.

HDAC (Histone Deacetylase) Inhibitor Valproic Acid Attenuates Atrial Remodeling and Delays the Onset of Atrial Fibrillation in Mice.

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.

Atrial fibrillation and heart failure-associated remodeling of two-pore-domain potassium (K2P) channels in murine disease models: focus on TASK-1.

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 (K2P) channels modulate cellular excitability, and TASK-1 (K2P3.1) 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 K2P channels and to assess modulation of K2P channel expression in murine models of AF and HF. Expression of cardiac K2P 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 K2P channel family, TASK-1 expression displayed highest levels in both atrial and ventricular tissue samples. Furthermore, K2P2.1, K2P5.1, and K2P6.1 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.

Stretch-activated two-pore-domain (K2P) potassium channels in the heart: Focus on atrial fibrillation and heart failure.

Two-pore-domain potassium (K2P) channels modulate cellular excitability. The significance of stretch-activated cardiac K2P channels (K2P2.1, TREK-1, KCNK2; K2P4.1, TRAAK, KCNK4; K2P10.1, 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 K2P channels in atrial fibrillation (AF) and heart failure (HF) patients in comparison to murine models. Cardiac K2P 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 K2P channel family (V > A). Disease-associated K2P2.1 remodeling was studied in detail. In patients with impaired left ventricular function, atrial KCNK2 (K2P2.1) mRNA and protein expression was significantly reduced. In AF subjects, downregulation of atrial and ventricular KCNK2 (K2P2.1) mRNA and protein levels was observed. AF-associated suppression of atrial Kcnk2 (K2P2.1) mRNA and protein was recapitulated in CREM-transgenic mice. Ventricular Kcnk2 expression was not significantly altered in mouse models of disease. In conclusion, mechanosensitive K2P2.1 and K2P10.1 K+ channels are expressed throughout the heart. HF- and AF-associated downregulation of KCNK2 (K2P2.1) mRNA and protein levels suggest a mechanistic contribution to cardiac arrhythmogenesis.

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 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 ß-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.

The European Network for Translational Research in Atrial Fibrillation (EUTRAF): objectives and initial results.

Atrial fibrillation (AF) is the most common sustained arrhythmia in the general population. As an age-related arrhythmia AF is becoming a huge socio-economic burden for European healthcare systems. Despite significant progress in our understanding of the pathophysiology of AF, therapeutic strategies for AF have not changed substantially and the major challenges in the management of AF are still unmet. This lack of progress may be related to the multifactorial pathogenesis of atrial remodelling and AF that hampers the identification of causative pathophysiological alterations in individual patients. Also, again new mechanisms have been identified and the relative contribution of these mechanisms still has to be established. In November 2010, the European Union launched the large collaborative project EUTRAF (European Network of Translational Research in Atrial Fibrillation) to address these challenges. The main aims of EUTRAF are to study the main mechanisms of initiation and perpetuation of AF, to identify the molecular alterations underlying atrial remodelling, to develop markers allowing to monitor this processes, and suggest strategies to treat AF based on insights in newly defined disease mechanisms. This article reports on the objectives, the structure, and initial results of this network.

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.

Neural peptidase endothelin-converting enzyme 1 regulates endothelin 1-induced pruritus.

In humans, pruritus (itch) is a common but poorly understood symptom in numerous skin and systemic diseases. Endothelin 1 (ET-1) evokes histamine-independent pruritus in mammals through activation of its cognate G protein-coupled receptor endothelin A receptor (ETAR). Here, we have identified neural endothelin-converting enzyme 1 (ECE-1) as a key regulator of ET-1-induced pruritus and neural signaling of itch. We show here that ETAR, ET-1, and ECE-1 are expressed and colocalize in murine dorsal root ganglia (DRG) neurons and human skin nerves. In murine DRG neurons, ET-1 induced internalization of ETAR within ECE-1-containing endosomes. ECE-1 inhibition slowed ETAR recycling yet prolonged ET-1-induced activation of ERK1/2, but not p38. In a murine itch model, ET-1-induced scratching behavior was substantially augmented by pharmacological ECE-1 inhibition and abrogated by treatment with an ERK1/2 inhibitor. Using iontophoresis, we demonstrated that ET-1 is a potent, partially histamine-independent pruritogen in humans. Immunohistochemical evaluation of skin from prurigo nodularis patients confirmed an upregulation of the ET-1/ETAR/ECE-1/ERK1/2 axis in patients with chronic itch. Together, our data identify the neural peptidase ECE-1 as a negative regulator of itch on sensory nerves by directly regulating ET-1-induced pruritus in humans and mice. Furthermore, these results implicate the ET-1/ECE-1/ERK1/2 pathway as a therapeutic target to treat pruritus in humans.

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.

Intravenous sphingosylphosphorylcholine protects ischemic and postischemic myocardial tissue in a mouse model of myocardial ischemia/reperfusion injury.

HDL, through sphingosine-1-phosphate (S1P), exerts direct cardioprotective effects on ischemic myocardium. It remains unclear whether other HDL-associated sphingophospholipids have similar effects. We therefore examined if HDL-associated sphingosylphosphorylcholine (SPC) reduces infarct size in a mouse model of transient myocardial ischemia/reperfusion. Intravenously administered SPC dose-dependently reduced infarct size after 30 minutes of myocardial ischemia and 24 hours reperfusion compared to controls. Infarct size was also reduced by postischemic, therapeutical administration of SPC. Immunohistochemistry revealed reduced polymorphonuclear neutrophil recruitment to the infarcted area after SPC treatment, and apoptosis was attenuated as measured by TUNEL. In vitro, SPC inhibited leukocyte adhesion to TNFα-activated endothelial cells and protected rat neonatal cardiomyocytes from apoptosis. S1P3 was identified as the lysophospholipid receptor mediating the cardioprotection by SPC, since its effect was completely absent in S1P3-deficient mice. We conclude that HDL-associated SPC directly protects against myocardial reperfusion injury in vivo via the S1P3 receptor.

Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice.

A trial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular Ca2+ handling. Diastolic Ca2+ release from the sarcoplasmic reticulum via "leaky" ryanodine receptors (RyR2s) is hypothesized to contribute to arrhythmogenesis in AF, but the molecular mechanisms are incompletely understood. Here, we have shown that mice with a genetic gain-of-function defect in Ryr2 (which we termed Ryr2R176Q/+ mice) did not exhibit spontaneous AF but that rapid atrial pacing unmasked an increased vulnerability to AF in these mice compared with wild-type mice. Rapid atrial pacing resulted in increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2, while both pharmacologic and genetic inhibition of CaMKII prevented AF inducibility in Ryr2R176Q/+ mice. This result suggests that AF requires both an arrhythmogenic substrate (e.g., RyR2 mutation) and enhanced CaMKII activity. Increased CaMKII phosphorylation of RyR2 was observed in atrial biopsies from mice with atrial enlargement and spontaneous AF, goats with lone AF, and patients with chronic AF. Genetic inhibition of CaMKII phosphorylation of RyR2 in Ryr2S2814A knockin mice reduced AF inducibility in a vagotonic AF model. Together, these findings suggest that increased RyR2-dependent Ca2+ leakage due to enhanced CaMKII activity is an important downstream effect of CaMKII in individuals susceptible to AF induction.

Critical role of transcription factor cyclic AMP response element modulator in beta1-adrenoceptor-mediated cardiac dysfunction.

BACKGROUND: Chronic stimulation of the beta(1)-adrenoceptor (beta(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 beta(1)AR-mediated cardiac deterioration. METHODS AND RESULTS: We studied the role of CREM in beta(1)AR-mediated cardiac effects, comparing transgenic mice with heart-directed expression of beta(1)AR in the absence and presence of functional CREM. CREM inactivation protected from cardiomyocyte hypertrophy, fibrosis, and left ventricular dysfunction in beta(1)AR-overexpressing mice. Transcriptome and proteome analysis revealed a set of predicted CREB/CREM target genes including the cardiac ryanodine receptor, tropomyosin 1alpha, and cardiac alpha-actin as altered on the mRNA or protein level along with the improved phenotype in CREM-deficient beta(1)AR-transgenic hearts. CONCLUSIONS: The results imply the regulation of genes by CREM as an important mechanism of beta(1)AR-induced cardiac damage in mice.

Nitric oxide synthase in muscular dystrophies: a re-evaluation.

Duchenne and Becker muscular dystrophies (DMD and BMD) are associated with decreased total nitric oxide (NO). However, mechanisms leading to NO deficiency with consequent muscle-cell degeneration remain unknown. To address this issue, we examined skeletal muscles of DMD and BMD patients for co-expression of NO synthase (NOS) with nitrotyrosine and transcription factor CREB, as well as with enzymes engaged in NO signaling. Employing immunocytochemical labeling, Western blotting and RT-PCR, we found that, in contrast to the most commonly accepted view, neuronal NOS was not restricted to the sarcolemma and that muscles of DMD and BMD patients retained all three NOS isoforms with an up-regulation of the inducible NOS isoform, CREB and nitrotyrosine. We suggest that enhanced nitrotyrosine immunostaining in muscle fibers as well as in the vasculature of DMD and BMD specimens reflects massive oxidative stress, resulting in withdrawal of NO from its regular physiological course via the scavenging actions of superoxides.