MAO-B-specific inhibition, either in knockout (KO) mice or pharmacologically, preserves left ventricular function and reduces cardiac fibrosis after myocardial infarction or pressure overload. We investigated whether stimulation of MAO-B in cardiac fibroblasts provokes ROS production and myofibroblast development. Fibroblast-specific MAO-B knockdown (KD) mice were created by crossing Col1a2CreERT mice with MAO-Bfl/fl mice. The KD was induced by tamoxifen injection. Fibroblasts of KD mice and wild types (WTs) were isolated and reduced MAO-B expression in KD fibroblasts was confirmed. In isolated mitochondria from the left ventricle of these mice, ROS production was reduced under stimulation with the specific MAO-B substrate β-phenylethylamine (PEA). Mitochondrial ROS production in fibroblasts, detected by MitoSox Red staining, increased under PEA (1000 µM) stimulation only in WT fibroblasts. mRNA of the marker genes for myofibroblast differentiation, Col1a1 and periostin, increased 2- or 3-fold, respectively, in WT but not in MAO-B KD fibroblasts. The enhanced migration potential under PEA was reduced in MAO-B KD fibroblasts. In conclusion, stimulation of MAO-B in cardiac fibroblasts leads to the formation of mitochondrial ROS, enhancement of myofibroblast marker gene expression and migration of the cells. Excessive fibrosis caused by elevated MAO-B activity in myocardial infarction can therefore contribute to cardiac dysfunction.

Intermediate monocytes (CD14++CD16+), a highly pro-inflammatory subset, are linked to endothelial activation, thrombus formation, and poor outcomes in acute coronary syndrome (ACS), suggesting a role in the transition to plaque vulnerability. MicroRNA-92a (miR-92a) promotes vascular inflammation by repressing the transcription factors Kruppel-like factors (KLFs) 2/4, thereby inducing endothelial dysfunction and increasing leukocyte adhesion. Because both intermediate monocytes and miR-92a contribute to plaque instability, their expression profiles appear relevant in acute ischemia. We investigated whether miR-92a is differentially regulated in monocyte subpopulations in ACS compared to chronic coronary syndrome (CCS). Patients with ACS (STEMI/NSTEMI) undergoing urgent coronary angiography and patients with CCS were enrolled. Blood samples were collected peripherally (T0P) and from the culprit coronary artery (T0C) during catheterization. Additional peripheral samples were collected 48 h after intervention (T1) and at the 3-month follow-up (T2). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density-gradient centrifugation. Monocytes were sorted by fluorescence-activated cell sorting (FACS) into classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) subsets. MiR-92a expression was measured using real-time PCR and analyzed across predefined time points. In classical and non-classical monocytes, miR-92a levels remained stable throughout the observation period and did not differ between ACS and CCS patients. No spatial expression gradient was observed between intracoronary and peripheral samples at baseline. In contrast, intermediate monocytes in the ACS cohort showed a transient increase in miR-92a expression at T1 compared with baseline (T0p) and the 3-month follow-up (T2). No comparable temporal changes were observed in CCS patients. These findings indicate a temporary alteration of miR-92a expression in intermediate monocytes during the early post-interventional phase following ACS. However, given the exploratory nature of this study and the limited sample size, the biological significance of this observation requires confirmation in larger cohorts.

Heart-specific overexpression of transcriptional regulator JDP2 (jun dimerization protein 2) for 5 weeks provokes paroxysmal atrial fibrillation (AF) in mice. We now investigated whether AF and atrial remodeling will be reversible upon termination of JDP2 overexpression, and whether paroxysmal AF converts to permanent AF in the presence of maintained JDP2 overexpression. Cardiac-specific JDP2 overexpression for 5 weeks, resulting in paroxysmal AF, was either continued or repressed via a tet-off system for another 5 weeks. ECGs were recorded weekly. Thereafter, heart and lung weights, and atrial mRNA and protein expression were determined. Extending JDP2 overexpression did not aggravate the AF phenotype, still paroxysmal AF, prolongation of PQ intervals, and atrial hypertrophy were present. This phenotype was completely reversible upon cessation of JDP2 overexpression. A massive downregulation of connexin40 and calcium handling proteins, including SERCA2a, calsequestrin, and ryanodine receptor, was observed in atria after prolonged JDP2 overexpression. In conclusion, atrial remodeling and paroxysmal AF under JDP2 overexpression are not sufficient to maintain or aggravate AF in the absence of JDP2. The comparison of the two groups indicates that the downregulation of calcium proteins and connexins is an important factor in the maintenance of the disease.

Atherosclerosis is a chronic proinflammatory disease of the vascular wall resulting in narrowing of arteries due to plaque formation, thereby causing reduced blood supply that is the leading cause for diverse end-organ damage with high mortality rates. Monocytes/macrophages, activated by elevated circulating lipoproteins, are significantly involved in the formation and development of atherosclerotic plaques. The imbalance between proinflammatory and anti-inflammatory macrophages, arising from dysregulated macrophage polarization, appears to be a driving force in this process. Proatherosclerotic processes acting on monocytes/macrophages include accumulation of cholesterol in macrophages leading to foam cell formation, as well as dysfunctional efferocytosis, all of which contribute to the formation of unstable plaques. In recent years, microRNAs (miRs) were identified as factors that could modulate monocyte/macrophage function and may therefore interfere with the atherosclerotic process. In this review, we present effects of monocyte/macrophage-derived miRs on atherosclerotic processes in order to reveal new treatment options using miRmimics or antagomiRs. image

Three types of highly promising small RNA therapeutics, namely, small interfering RNAs (siRNAs), microRNAs (miRNAs) and the RNA subtype of antisense oligonucleotides (ASOs), offer advantages over small-molecule drugs. These small RNAs can target any gene product, opening up new avenues of effective and safe therapeutic approaches for a wide range of diseases. In preclinical research, synthetic small RNAs play an essential role in the investigation of physiological and pathological pathways as silencers of specific genes, facilitating discovery and validation of drug targets in different conditions. Off-target effects of small RNAs, however, could make it difficult to interpret experimental results in the preclinical phase and may contribute to adverse events of small RNA therapeutics. Out of the two major types of off-target effects we focused on the hybridization-dependent, especially on the miRNA-like off-target effects. Our main aim was to discuss several approaches, including sequence design, chemical modifications and target prediction, to reduce hybridization-dependent off-target effects that should be considered even at the early development phase of small RNA therapy. Because there is no standard way of predicting hybridization-dependent off-target effects, this review provides an overview of all major state-of-the-art computational methods and proposes new approaches, such as the possible inclusion of network theory and artificial intelligence (AI) in the prediction workflows. Case studies and a concise survey of experimental methods for validating in silico predictions are also presented. These methods could contribute to interpret experimental results, to minimize off-target effects and hopefully to avoid off-target-related adverse events of small RNA therapeutics. LINKED ARTICLES: This article is part of a themed issue Non-coding RNA Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.2/issuetoc.

Cardiac hypertrophy resulting from sympathetic nervous system activation triggers the development of heart failure. The transcription factor Y-box binding protein 1 (YB-1) can interact with transcription factors involved in cardiac hypertrophy and may thereby interfere with the hypertrophy growth process. Therefore, the question arises as to whether YB-1 influences cardiomyocyte hypertrophy and might thereby influence the development of heart failure. YB-1 expression is downregulated in human heart biopsies of patients with ischemic cardiomyopathy (n = 8), leading to heart failure. To study the impact of reduced YB-1 in cardiac cells, we performed small interfering RNA (siRNA) experiments in H9C2 cells as well as in adult cardiomyocytes (CMs) of rats. The specificity of YB-1 siRNA was analyzed by a miRNA-like off-target prediction assay identifying potential genes. Testing three high-scoring genes by transfecting cardiac cells with YB-1 siRNA did not result in downregulation of these genes in contrast to YB-1, whose downregulation increased hypertrophic growth. Hypertrophic growth was mediated by PI3K under PE stimulation, as well by downregulation with YB-1 siRNA. On the other hand, overexpression of YB-1 in CMs, caused by infection with an adenovirus encoding YB-1 (AdYB-1), prevented hypertrophic growth under α-adrenergic stimulation with phenylephrine (PE), but not under stimulation with growth differentiation factor 15 (GDF15; n = 10–16). An adenovirus encoding the green fluorescent protein (AdGFP) served as the control. YB-1 overexpression enhanced the mRNA expression of the Gq inhibitor regulator of G-protein signaling 2 (RGS2) under PE stimulation (n = 6), potentially explaining its inhibitory effect on PE-induced hypertrophic growth. This study shows that YB-1 protects cardiomyocytes against PE-induced hypertrophic growth. Like in human end-stage heart failure, YB-1 downregulation may cause the heart to lose its protection against hypertrophic stimuli and progress to heart failure. Therefore, the transcription factor YB-1 is a pivotal signaling molecule, providing perspectives for therapeutic approaches.

Cardiac and vascular diseases are often associated with increased oxidative stress and inflammation, and both may contribute to the disease progression. However, successful applications of antioxidants in the clinical setting are very rare and specific anti-inflammatory therapeutics only emerged recently. Reasons for this rely on the great diversity of oxidative stress and inflammatory cells that can either act as cardioprotective or cause tissue damage in the heart. Recent large-scale clinical trials found that highly specific anti-inflammatory therapies using monoclonal antibodies against cytokines resulted in lower cardiovascular mortality in patients with pre-existing atherosclerotic disease. In addition, unspecific antiinflammatory medication and established cardiovascular drugs with pleiotropic immunomodulatory properties such as angiotensin converting enzyme (ACE) inhibitors or statins have proven beneficial cardiovascular effects. Normalization of oxidative stress seems to be a common feature of these therapies, which can be explained by a close interaction/crosstalk of the cellular redox state and inflammatory processes. In this review, we give an overview of cardiac reactive oxygen species (ROS) sources and processes of cardiac inflammation as well as the connection of ROS and inflammation in ischemic cardiomyopathy in order to shed light on possible cardioprotective interventions.

Endometrial cancer (EC) has been associated with an increased risk of cardiovascular disease, including atrial fibrillation (AF). We performed a prospective, case-controlled analysis among 310 Bulgarian women with new-onset, histologically confirmed EC, free of AF at the baseline survey, and women with normal (senile) endometrium/endometrial hyperplasia as a control group (n = 205). The risk of AF as well as relationship of adiponectin (APN) and high sensitivity C-reactive protein (hs-CRP) levels with AF in women with EC were calculated by Cox proportional hazards models. During the mean follow-up of 2.5 ± 0.5 years, new-onset AF had occurred in 11.7% of women with EC vs. 5.8% in the control group (p < 0.01). The risk of AF was highest in the first 6 months after new-onset EC, with an incidence rate ratio (IRR) of 1.19 (95% CI 1.10–1.29; p = 0.01). Women with EC, who were obese (body mass index (BMI) > 30 kg/m2) and younger (age < 60) were found to be more likely to develop AF (HR 1.95; 95% CI 1.18–3.32; p = 0.05). APN levels were not significantly associated with new-onset AF (95% CI 0.87–1.21; p = 0.063). However, the secondary analysis showed evidence of APN–AF association when adjusted for BMI (2.05; 95% CI 1.04–4.04; p = 0.037). We conclude that EC was significantly associated with the incidence of AF.

Cardiac and vascular diseases are often associated with increased oxidative stress and inflammation, and both may contribute to the disease progression. However, successful applications of antioxidants in the clinical setting are very rare and specific anti-inflammatory therapeutics only emerged recently. Reasons for this rely on the great diversity of oxidative stress and inflammatory cells that can either act as cardioprotective or cause tissue damage in the heart. Recent large-scale clinical trials found that highly specific anti-inflammatory therapies using monoclonal antibodies against cytokines resulted in lower cardiovascular mortality in patients with pre-existing atherosclerotic disease. In addition, unspecific antiinflammatory medication and established cardiovascular drugs with pleiotropic immunomodulatory properties such as angiotensin converting enzyme (ACE) inhibitors or statins have proven beneficial cardiovascular effects. Normalization of oxidative stress seems to be a common feature of these therapies, which can be explained by a close interaction/crosstalk of the cellular redox state and inflammatory processes. In this review, we give an overview of cardiac reactive oxygen species (ROS) sources and processes of cardiac inflammation as well as the connection of ROS and inflammation in ischemic cardiomyopathy in order to shed light on possible cardioprotective interventions.

Background: TGFβ1 is a growth factor that plays a major role in the remodeling process of the heart by inducing cardiomyocyte dysfunction and apoptosis, as well as fibrosis thereby restricting heart function. TGFβ1 mediates its effect via the TGFβ receptor I (ALK5) and the activation of SMAD transcription factors, but TGFβ1 is also known as activator of phosphoinositide-3-kinase (PI3K) via the non-SMAD signaling pathway. The aim of this study was to investigate whether PI3K is also involved in TGFβ1–induced cardiomyocytes apoptosis and contractile dysfunction. Methods and Results: Incubation of isolated ventricular cardiomyocytes with TGFβ1 resulted in impaired contractile function. Pre-incubation of cells with the PI3K inhibitor Ly294002 or the ALK5 inhibitor SB431542 attenuated the decreased cell shortening in TGFβ1–stimulated cells. Additionally, TGFβ-induced apoptosis was significantly reduced by the PI3K inhibitor Ly294002. Administration of a PI3Kγ-specific inhibitor AS605240 abolished the TGFβ effect on apoptosis and cell shortening. This was also confirmed in cardiomyocytes from PI3Kγ KO mice. Induction of SMAD binding activity and the TGFβ target gene collagen 1 could be blocked by the PI3K inhibitor Ly294002, but not by the specific PI3Kγ inhibitor AS605240. Conclusions: TGFβ1-induced SMAD activation, cardiomyocyte apoptosis, and impaired cell shortening are mediated via both, the ALK5 receptor and PI3K, in adult cardiomyocytes. PI3Kγ specifically contributes to apoptosis induction and impairment of contractile function independent of SMAD signaling.

Reperfusion is the only feasible therapy following myocardial infarction, but reperfusion has been shown to damage mitochondrial function and disrupt energy production in the heart. Adenine nucleotide translocase 1 (ANT1) facilitates the transfer of ADP/ATP across the inner mitochondrial membrane; therefore, we tested whether ANT1 exerts protective effects on mitochondrial function during ischemia/reperfusion (I/R). The hearts of wild-type (WT) and transgenic ANT1-overexpressing (ANT1-TG) rats were exposed to I/R injury using the standard Langendorff technique, after which mitochondrial function, hemodynamic parameters, infarct size, and components of the contractile apparatus were determined. ANT1-TG hearts expressed higher ANT protein levels, with reduced levels of oxidative 4-hydroxynonenal ANT modifications following I/R. ANT1-TG mitochondria isolated from I/R hearts displayed stable calcium retention capacity (CRC) and improved membrane potential stability compared with WT mitochondria. Mitochondria isolated from ANT1-TG hearts experienced less restricted oxygen consumption than WT mitochondria after I/R. Left ventricular diastolic pressure (Pdia) decreased in ANT1-TG hearts compared with WT hearts following I/R. Preserved diastolic function was accompanied by a decrease in the phospho-lamban (PLB)/sarcoplasmic reticulum calcium ATPase (SERCA2a) ratio in ANT1-TG hearts compared with that in WT hearts. In addition, the phosphorylated (P)-PLB/PLB ratio increased in ANT1-TG hearts after I/R but not in WT hearts, which indicated more effective calcium uptake into the sarcoplasmic reticulum in ANT1-TG hearts. In conclusion, ANT1-TG rat hearts coped more efficiently with I/R than WT rat hearts, which was reflected by preserved mitochondrial energy balance, diastolic function, and calcium dynamics after reperfusion.

Matrix metalloproteinases (MMPs) are identified as modulators of the extracellular matrix in heart failure progression. However, evidence for intracellular effects of MMPs is emerging. Pro- and anti-hypertrophic cardiac effects are described. This may be due to the various sources of different MMPs in the heart tissue. Therefore, the aim of the present study was to determine the role of MMPs in hypertrophic growth of isolated rat ventricular cardiac myocytes. Cardiomyocytes were isolated form ventricular tissues of the rat hearts by collagenase perfusion. RT-qPCR, western blots, and zymography were used for expression and MMP activity analysis. Cross-sectional area and the rate of protein synthesis were determined as parameters for hypertrophic growth. MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 mRNAs were detected in cardiomyocytes, and protein expression of MMP-2, MMP-9, and MMP-14 was identified. Hypertrophic stimulation of cardiomyocytes did not enhance, but interestingly decreased expression of MMPs, indicating that downregulation of MMPs may promote hypertrophic growth. Indeed, the nonselective MMP inhibitors TAPI-0 or TIMP2 and the MMP-2-selective ARP-100 enhanced hypertrophic growth. Furthermore, TAPI-0 increased phosphorylation and thus activation of extracellular signaling kinase (ERK) and Akt (protein kinase B), as well as inhibition of glycogen synthase 3β (GSK3β). Abrogation of MEK/ERK- or phosphatidylinositol-3-kinase(PI3K)/Akt/GSK3β-signaling with PD98059 or LY290042, respectively, inhibited hypertrophic growth under TAPI-0. MMPs’ inhibition promotes hypertrophic growth in cardiomyocytes in vitro. Therefore, MMPs in the healthy heart may be important players to repress cardiac hypertrophy.

Heart failure (HF) and atrial fibrillation (AF) are two major life-threatening diseases worldwide. Causes and mechanisms are incompletely understood, yet current therapies are unable to stop disease progression. In this review, we focus on the contribution of the transcriptional modulator, Jun dimerization protein 2 (JDP2), and on HF and AF development. In recent years, JDP2 has been identified as a potential prognostic marker for HF development after myocardial infarction. This close correlation to the disease development suggests that JDP2 may be involved in initiation and progression of HF as well as in cardiac dysfunction. Although no studies have been done in humans yet, studies on genetically modified mice impressively show involvement of JDP2 in HF and AF, making it an interesting therapeutic target.

BACKGROUND:Cardiac-specific JDP2 overexpression provokes ventricular dysfunction and atrial dilatation in mice. We performed in vivo studies on JDP2-overexpressing mice to investigate the impact of JDP2 on the predisposition to spontaneous atrial fibrillation (AF). METHODS:JDP2-overexpression was started by withdrawal of a doxycycline diet in 4-week-old mice. The spontaneous onset of AF was documented by ECG within 4 to 5 weeks of JDP2 overexpression. Gene expression was analyzed by real-time RT-PCR and Western blots. RESULTS:In atrial tissue of JDP2 mice, besides the 3.6-fold increase of JDP2 mRNA, no changes could be detected within one week of JDP2 overexpression. Atrial dilatation and hypertrophy, combined with elongated cardiomyocytes and fibrosis, became evident after 5 weeks of JDP2 overexpression. Electrocardiogram (ECG) recordings revealed prolonged PQ-intervals and broadened P-waves and QRS-complexes, as well as AV-blocks and paroxysmal AF. Furthermore, reductions were found in the atrial mRNA and protein level of the calcium-handling proteins NCX, Cav1.2 and RyR2, as well as of connexin40 mRNA. mRNA of the hypertrophic marker gene ANP, pro-inflammatory MCP1, as well as markers of immune cell infiltration (CD68, CD20) were increased in JDP2 mice. CONCLUSION:JDP2 is an important regulator of atrial calcium and immune homeostasis and is involved in the development of atrial conduction defects and arrhythmogenic substrates preceding paroxysmal AF.

Background Factor VII activating protease (FSAP) is of interest as a marker for vascular inflammation and plaque destabilization. The aim of this study was to analyze the expression profile of FSAP in endarterectomy specimens that were taken from patients with asymptomatic and symptomatic carotid atherosclerotic plaques and to compare them with circulating FSAP levels. Methods and Results Plasma FSAP concentration, activity, and mRNA expression were measured in endarterectomy specimens and in monocytes and platelets. Plaque and plasma FSAP levels were higher in symptomatic patients (n=10) than in asymptomatic patients (n=14). Stronger FSAP immunostaining was observed in advanced symptomatic lesions, in intraplaque hemorrhage-related structures, and in lipid-rich areas within the necrotic core. FSAP was also colocalized with monocytes and macrophages (CD11b/CD68-positive cells) and platelets (CD41-positive cells) of the plaques. Moreover, human platelets expressed FSAP in vitro, at both the mRNA and protein levels. Expression is stimulated by thrombin receptor-activating peptide and ADP and reduced by acetylsalicylic acid. Conclusions Plasma FSAP levels were significantly increased in patients with symptomatic carotid stenosis and thus may be involved in plaque development This plaque-associated FSAP may be produced by platelets or macrophages or may be taken up from the circulation. To establish FSAP's utility as a circulating or plaque biomarker in patients with symptomatic carotid atherosclerotic plaques, further studies are needed.

When you think about hypoxia-inducible factor-1 (HIF-1), what comes in your mind first? Nearly everyone will answer something like ‘It’s an oxygen-sensitive transcriptional regulator that protects cells against damage under oxygen deprivation’. Indeed, HIF-1 is a dimer that is made up of the constitutively expressed HIF-1b and the oxygen labile subunit HIF-1a, first described by Semenza and Wang. In the presence of oxygen, hydroxylation of HIF-1a by the prolyl-4-hydroxylase domain-containing enzyme (PHD) targets HIF-1a for ubiquitination and proteasomal degradation. Besides this regulation of HIF-1a stability, in 2001 another regulatory step was described, namely the reduction of HIF-1a transcriptional activity by the factor inhibiting (FIH-1) (Figure 1). Also, this is an oxygen-sensitive process which leads to increased HIF activity under oxygen deficiency. Due to the exclusive presence and activation of HIF-1 only under hypoxic conditions, this transcriptional regulator is an ideal factor in the fight against ischaemic tissue injury. In fact, early research about HIF-1 focused on this issue and identified lots of protective mechanisms. HIF-1 binds to hypoxia responsive element (HRE) in the promoter of many genes among which erythropoietin is responsible for erythrocyte synthesis or VEGF (vascular endothelial growth factor) that promotes angiogenesis. Therefore, the presence of HIF-1 in an oxygen-reduced area tries to counteract this miserable situation by improving the transport of oxygen via an increased number of vessels supplying this area, via enhancement of the number of erythrocytes that transport the oxygen, and via a switch from oxidative to glycolytic metabolism. In heart research, HIF-1 also moved into the interest of researchers due to the fact that myocardial infarction still is one of the most frequent causes of death, and the search for new therapies is ongoing. At the beginning of this century, reports came up showing infarct size reduction upon enhancement of HIF-1a expression. Also, involvement of HIF-1a in ischaemic preconditioning was shown. In transgenic mice with partial inhibition of HIF-1a acute and delayed preconditioning was abolished. Even the remodelling process after myocardial infarction could be reduced by PHD inhibitors when applied for some days or weeks. However, the long-term stabilization of HIF-1 in transgenic mice by PHD inactivation or HIF-1 stabilization provoked dilated cardiomyopathy and contractile dysfunction. This is in line with the increased HIF-1 levels found in heart failure patients. Thus, in the acute ischaemic situation HIF-1 induction helps to reduce infarct size, whereas upon long-term stabilization of HIF-1 the effects are reversed and become detrimental. Differences in the HIF responsiveness may be caused by genetic and/ or epigenetic modifications. While HIF target genes are up-regulated under hypoxia, common transcription in the cell is largely down-regulated. These variations in gene expression under hypoxic conditions are related to multiple post-translational modifications of histones (H3, H4) like acetylation or methylation of lysine that can result either in transcriptional activation or repression. The transcriptional responses to HIF-1 are context-dependent which means that different cell types show diverse patterns of gene expression upon HIF-1 stimulation, and can be traced back to different arrangements of histone modifications in their promotors or enhancers. For example, H3K9ac and H3K4me3 levels are increased in the VEGF promotor after HIF induction that recruits a series of gene-specific factors. In this regard, Schunke et al. identified PRKCBP1 (also called RACK7 or ZMYND8) as a new oxygen-dependent regulator of HIF in the heart that was so far only investigated in cancer. In breast cancer the epigenetic reader PRKCBP1 is recruited among others by H3K14ac and interacts with the HIF complex. During this process p300 acetylates PRKCBP1, which stimulates RNA polymerase II phosphorylation resulting in HIFdependent gene transcription. In contrast, the recognition of the combined histone modification mark H3K4me1 and H3K14ac by PRKCBP1 results in suppression of expression in cancer cells likely by histone demethylation of H3K4me1. PRKCBP1 induction in the heart was found by accident. It was a mouse strain-specific effect since only part of the transgenic animals (C57BL/6) with stable HIF-1 expression responded adequately with positive angiogenic effects as well as with ventricular dysfunction. Schunke et al. decided to have a closer look at the nonresponsive animals (FVB/N), and detected via CHIP analysis that 78% of the HIF-1 bound sequences belong to the PRKCBP1 gene. This high binding affinity was caused by a genetic DNA modification, namely an insertion of two canonical HRE-sites in front of the PRKCBP1 gene in FVB mice only. The induction of PRKCBP1 in mice strain subpopulations may then cause epigenetic effects resulting in decreased enhancer activities, reduced HIF actions, and loss of the typical phenotype, normally apparent under stable HIF expression, thereby acting as negative feedback loop on HIF-1 (Figure 1).

Abstract Introduction JDP2 (Jun Dimerization Protein 2) was recently characterized as potential prognostic marker for progression of heart failure after myocardial infarction. Furthermore, persistent cardiac-specific JDP2 overexpression provokes ventricular dysfunction, atrial dilatation and atrial fibrillation. Purpose In this study we investigated, if ventricular dysfunction is predisposed to atrial fibrillation. Therefore, we analyzed ventricular function in mice with 1 and 5 weeks of JDP2 overexpression and compared gene expression in ventricular and atrial tissues at these time points. Methods JDP2 expression was under control of a Tet-off system. Therefore, JDP2 overexpression was started by withdrawal of doxycycline diet in 4-week-old mice. After 1 or 5 weeks, cardiac function was determined by echocardiography and ECG. mRNA expression was analyzed by real-time RT-PCR and protein expression in western blots. Results After 1 or 5 weeks, JDP2 mRNA levels were increased in ventricular and atrial tissues of JDP2 mice. Already after 1 week ventricular dysfunction emerged in JDP2 mice: Ejection fraction decreased from 64.6±10.4% in WT to 58.8±9.3% in JDP2 mice, fractional shortening from 38.3±7.9% in WT to 27.4±4.8% in JDP -mice, and cardiac output from 23.0±4.7 ml/min in WT to 19.4±3.3 ml/min in JDP2 mice (n=11–16, p<0.05). In ventricular tissues, elastin mRNA expression increased, and the calcium handling protein SERCA decreased within one week of JDP2 overexpression (n=6, p<0.05 vs. WT). After 5 weeks of JDP2 overexpression ventricular dysfunction became even stronger with a cardiac output of 13.6±2.5 ml/min (n=11, p<0.05 vs. WT). Still reduction in SERCA protein was observed, and increased mRNA levels of fibrotic marker genes were detected, as well as contractile function of isolated cardiomyocytes of JDP2 mice continued to decline. In atrial tissue, besides the 3.6 times increase of JDP2 mRNA, no changes could be detected within one week. In addition, ECG recordings over 30 minutes on awake mice did not show any abnormalities after one week of JDP2-overexpression. Atrial dilatation became evident only after 5 weeks of JDP2 overexpression. ECG-recordings revealed prolonged PR-intervals (34.6±3.9 ms in JDP2-mice vs. 30.2±7.0 ms in WT) and broadened QRS-complexes (15.8±2.8 ms in JDP2 mice vs. 13.9±1.7 ms in WT, n=10–12, p<0.05). At the same time point, mRNA of the calcium-handling proteins PLB, NCX and SERCA, and of the fibrotic marker genes collagen I, fibronectin and elastin were dramatically reduced in atrial tissue of JDP2 compared to WT mice (n=6, p<0.05), and may be functionally involved in atrial conduction defects. Conclusion Enhanced expression of JDP2 provokes ventricular dysfunction and fibrosis within one week, whereas changes in the atrial tissue and conduction defects occur later and seem to be a secondary effect that is provoked by the pre-existing ventricular dysfunction.

The transcriptional regulator JDP2 (Jun dimerization protein 2) has been identified as a prognostic marker for patients to develop heart failure after myocardial infarction. We now performed in vivo studies on JDP2-overexpressing mice, to clarify the impact of JDP2 on heart failure progression. Therefore, during birth up to the age of 4 weeks cardiac-specific JDP2 overexpression was prevented by doxycycline feeding in transgenic mice. Then, JDP2 overexpression was started. Already after 1 week, cardiac function, determined by echocardiography, decreased which was also resembled on the cardiomyocyte level. After 5 weeks blood pressure declined, ejection fraction and cardiac output was reduced and left ventricular dilatation developed. Heart weight/body weight, and mRNA expression of ANP, inflammatory marker genes, collagen and fibronectin increased. Collagen 1 protein expression increased, and fibrosis developed. As an additional sign of elevated extracellular matrix remodeling, matrix metalloproteinase 2 activity increased in JDP2 mice. Thus, JDP2 overexpression is deleterious to heart function in vivo. It can be concluded that JDP2 overexpression provokes cardiac dysfunction in adult mice that is accompanied by hypertrophy and fibrosis. Thus, induction of JDP2 is a maladaptive response contributing to heart failure development.

MicroRNA (miR) is reported to be involved in vascular inflammation and may represent a novel class of diagnostic biomarkers in cardiovascular disease. We aimed to identify the miR expression profile in human advanced coronary atherosclerotic plaques (CAP) and to connect this expression to the processes in atherosclerosis. Microarray techniques and TaqMan polymerase chain reaction were used to analyse the global expression of 352 miRs in CAP obtained during ACS MULTI-LINK study. 11 miRs were selected on the basis of their implication in atherosclerosis, endothelial activation, and inflammation. 6 miRs were found to be differently expressed in CAP when compared to non-atherosclerotic internal mammary arteries (IMA, p < 0.05). The expression of miR-21, -92a, and -99a was verified and found to be significantly up-regulated in CAP versus IMA (p < 0.001). We also performed bioinformatic analysis and found several potential target genes of miR-92a and -99a as well as several pathways with impact on atherosclerosis which could be differently expressed due to this miRNA profile. The most up-regulated miRs are involved in processes known to be connected to atherosclerosis. Interfering with the miR expression in the artery wall is a potential way to affect atherosclerotic plaque and cardiovascular disease development.