Stefan Engelhardt

MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts.

MicroRNAs comprise a broad class of small non-coding RNAs that control expression of complementary target messenger RNAs. Dysregulation of microRNAs by several mechanisms has been described in various disease states including cardiac disease. Whereas previous studies of cardiac disease have focused on microRNAs that are primarily expressed in cardiomyocytes, the role of microRNAs expressed in other cell types of the heart is unclear. Here we show that microRNA-21 (miR-21, also known as Mirn21) regulates the ERK–MAP kinase signalling pathway in cardiac fibroblasts, which has impacts on global cardiac structure and function. miR-21 levels are increased selectively in fibroblasts of the failing heart, augmenting ERK–MAP kinase activity through inhibition of sprouty homologue 1 (Spry1). This mechanism regulates fibroblast survival and growth factor secretion, apparently controlling the extent of interstitial fibrosis and cardiac hypertrophy. In vivo silencing of miR-21 by a specific antagomir in a mouse pressure-overload-induced disease model reduces cardiac ERK–MAP kinase activity, inhibits interstitial fibrosis and attenuates cardiac dysfunction. These findings reveal that microRNAs can contribute to myocardial disease by an effect in cardiac fibroblasts. Our results validate miR-21 as a disease target in heart failure and establish the therapeutic efficacy of microRNA therapeutic intervention in a cardiovascular disease setting.

MicroRNAs in the human heart : A clue to fetal gene reprogramming in heart failure

Background— Chronic heart failure is characterized by left ventricular remodeling and reactivation of a fetal gene program; the underlying mechanisms are only partly understood. Here we provide evidence that cardiac microRNAs, recently discovered key regulators of gene expression, contribute to the transcriptional changes observed in heart failure. Methods and Results— Cardiac transcriptome analyses revealed striking similarities between fetal and failing human heart tissue. Using microRNA arrays, we discovered profound alterations of microRNA expression in failing hearts. These changes closely mimicked the microRNA expression pattern observed in fetal cardiac tissue. Bioinformatic analysis demonstrated a striking concordance between regulated messenger RNA expression in heart failure and the presence of microRNA binding sites in the respective 3 untranslated regions. Messenger RNAs upregulated in the failing heart contained preferentially binding sites for downregulated microRNAs and vice versa. Mechanistically, transfection of cardiomyocytes with a set of fetal microRNAs induced cellular hypertrophy as well as changes in gene expression comparable to the failing heart. Conclusions— Our data support a novel mode of regulation for the transcriptional changes in cardiac failure. Reactivation of a fetal microRNA program substantially contributes to alterations of gene expression in the failing human heart.

What Is the Role of β-Adrenergic Signaling in Heart Failure?

Abstract— This review addresses open questions about the role of &bgr;-adrenergic receptors in cardiac function and failure. Cardiomyocytes express all three &bgr;-adrenergic receptor subtypes—&bgr;1, &bgr;2, and, at least in some species, &bgr;3. The &bgr;1 subtype is the most prominent one and is mainly responsible for positive chronotropic and inotropic effects of catecholamines. The &bgr;2 subtype also increases cardiac function, but its ability to activate nonclassical signaling pathways suggests a function distinct from the &bgr;1 subtype. In heart failure, the sympathetic system is activated, cardiac &bgr;-receptor number and function are decreased, and downstream mechanisms are altered. However, in spite of a wealth of data, we still do not know whether and to what extent these alterations are adaptive/protective or detrimental, or both. Clinically, &bgr;-adrenergic antagonists represent the most important advance in heart failure therapy, but it is still debated whether they act by blocking or by resensitizing the &bgr;-adrenergic receptor system. Newer experimental therapeutic strategies aim at the receptor desensitization machinery and at downstream signaling steps.

MicroRNA-24 Regulates Vascularity After Myocardial Infarction

Background— Myocardial infarction leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. Methods and Results— Here, we show that the small noncoding RNA microRNA-24 (miR-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. MiR-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on Matrigel, and inhibits cell sprouting from endothelial spheroids. These effects are mediated through targeting of the endothelium-enriched transcription factor GATA2 and the p21-activated kinase PAK4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. Respective downstream signaling cascades involving phosphorylated BAD (Bcl-XL/Bcl-2–associated death promoter) and Sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blocking of endothelial miR-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival. Conclusions— Our findings indicate that miR-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesis and is suitable for therapeutic intervention in the setting of ischemic heart disease.

Cyclic AMP Imaging in Adult Cardiac Myocytes Reveals Far-Reaching β1-Adrenergic but Locally Confined β2-Adrenergic Receptor–Mediated Signaling

&bgr;1- and &bgr;2-adrenergic receptors (&bgr;ARs) are known to differentially regulate cardiomyocyte contraction and growth. We tested the hypothesis that these differences are attributable to spatial compartmentation of the second messenger cAMP. Using a fluorescent resonance energy transfer (FRET)-based approach, we directly monitored the spatial and temporal distribution of cAMP in adult cardiomyocytes. We developed a new cAMP-FRET sensor (termed HCN2-camps) based on a single cAMP binding domain of the hyperpolarization activated cyclic nucleotide-gated potassium channel 2 (HCN2). Its cytosolic distribution, high dynamic range, and sensitivity make HCN2-camps particularly well suited to monitor subcellular localization of cardiomyocyte cAMP. We generated HCN2-camps transgenic mice and performed single-cell FRET imaging on freshly isolated cardiomyocytes. Whole-cell superfusion with isoproterenol showed a moderate elevation of cAMP. Application of various phosphodiesterase (PDE) inhibitors revealed stringent control of cAMP through PDE4>PDE2>PDE3. The &bgr;1AR-mediated cAMP signals were entirely dependent on PDE4 activity, whereas &bgr;2AR-mediated cAMP was under control of multiple PDE isoforms. &bgr;1AR subtype–specific stimulation yielded ≈2-fold greater cAMP responses compared with selective &bgr;2-subtype stimulation, even on treatment with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (&Dgr;FRET, 17.3±1.3% [&bgr;1AR] versus 8.8±0.4% [&bgr;2AR]). Treatment with pertussis toxin to inactivate Gi did not affect cAMP production. Localized &bgr;1AR stimulation generated a cAMP gradient propagating throughout the cell, whereas local &bgr;2AR stimulation did not elicit marked cAMP diffusion. Our data reveal that in adult cardiac myocytes, &bgr;1ARs induce far-reaching cAMP signals, whereas &bgr;2AR-induced cAMP remains locally confined.

The miRNA-212/132 family regulates both cardiac hypertrophy and cardiomyocyte autophagy

Pathological growth of cardiomyocytes (hypertrophy) is a major determinant for the development of heart failure, one of the leading medical causes of mortality worldwide. Here we show that the microRNA (miRNA)-212/132 family regulates cardiac hypertrophy and autophagy in cardiomyocytes. Hypertrophic stimuli upregulate cardiomyocyte expression of miR-212 and miR-132, which are both necessary and sufficient to drive the hypertrophic growth of cardiomyocytes. MiR-212/132 null mice are protected from pressure-overload-induced heart failure, whereas cardiomyocyte-specific overexpression of the miR-212/132 family leads to pathological cardiac hypertrophy, heart failure and death in mice. Both miR-212 and miR-132 directly target the anti-hypertrophic and pro-autophagic FoxO3 transcription factor and overexpression of these miRNAs leads to hyperactivation of pro-hypertrophic calcineurin/NFAT signalling and an impaired autophagic response upon starvation. Pharmacological inhibition of miR-132 by antagomir injection rescues cardiac hypertrophy and heart failure in mice, offering a possible therapeutic approach for cardiac failure.

Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1

Fungal and oomycete plant pathogens translocate effector proteins into host cells to establish infection. However, virulence targets and modes of action of their effectors are unknown. Effector AVR3a from potato blight pathogen Phytophthora infestans is translocated into host cells and occurs in two forms: AVR3aKI, which is detected by potato resistance protein R3a, strongly suppresses infestin 1 (INF1)-triggered cell death (ICD), whereas AVR3aEM, which evades recognition by R3a, weakly suppresses host ICD. Here we show that AVR3a interacts with and stabilizes host U-box E3 ligase CMPG1, which is required for ICD. In contrast, AVR3aKI/Y147del, a mutant with a deleted C-terminal tyrosine residue that fails to suppress ICD, cannot interact with or stabilize CMPG1. CMPG1 is stabilized by the inhibitors MG132 and epoxomicin, indicating that it is degraded by the 26S proteasome. CMPG1 is degraded during ICD. However, it is stabilized by mutations in the U-box that prevent its E3 ligase activity. In stabilizing CMPG1, AVR3a thus modifies its normal activity. Remarkably, given the potential for hundreds of effector genes in the P. infestans genome, silencing Avr3a compromises P. infestans pathogenicity, suggesting that AVR3a is essential for virulence. Interestingly, Avr3a silencing can be complemented by in planta expression of Avr3aKI or Avr3aEM but not the Avr3aKI/Y147del mutant. Our data provide genetic evidence that AVR3a is an essential virulence factor that targets and stabilizes the plant E3 ligase CMPG1, potentially to prevent host cell death during the biotrophic phase of infection.

Inhibition of Na+-H+ Exchange Prevents Hypertrophy, Fibrosis, and Heart Failure in β1-Adrenergic Receptor Transgenic Mice

Chronic stimulation of the &bgr;1-adrenergic receptor leads to hypertrophy and heart failure in &bgr;1-adrenergic receptor transgenic mice and contributes to disease progression in heart failure patients. The cellular mechanisms underlying these detrimental effects are largely unknown. In this study, we have identified the cardiac Na+-H+ exchanger (NHE1) as a novel mediator of adrenergically induced heart failure. &bgr;1-Adrenergic receptor transgenic mice showed upregulation of both NHE1 mRNA (+140±6%) and protein (+42±19%). In order to test whether increased NHE1 is causally related to &bgr;1-adrenergic–induced hypertrophy, fibrosis, and heart failure, &bgr;1-adrenergic receptor transgenic (TG) and wild-type (WT) littermates were treated with a diet containing 6000 ppm of the NHE1 inhibitor cariporide or control chow for 8 months. There was significant hypertrophy of cardiac myocytes in &bgr;1-adrenergic receptor transgenic mice (2.3-fold increase in myocyte cross-sectional area), which was virtually absent in cariporide-fed animals. Interstitial fibrosis was prominent throughout the left ventricular wall in nontreated &bgr;1-adrenergic receptor transgenic mice (4.8-fold increase in collagen volume fraction); cariporide treatment completely prevented this development of fibrosis. Left ventricular catheterization showed that cariporide also prevented the loss of contractile function in &bgr;1-adrenergic receptor transgenic mice: whereas untreated transgenic mice showed a significant decrease in left ventricular contractility (5250±570 mm Hg/s TG versus 7360±540 mm Hg/s WT, dp/dtmax), this decrease was completely prevented by cariporide (8150±520 mm Hg/s TG cariporide). Inhibition of NHE1 prevented the development of heart failure in &bgr;1-receptor transgenic mice. We conclude that the cardiac Na+-H+ exchanger 1 is essential for the detrimental cardiac effects of chronic &bgr;1-receptor stimulation in the heart.

Dobutamine-Stress Magnetic Resonance Microimaging in Mice Acute Changes of Cardiac Geometry and Function in Normal and Failing Murine Hearts

Abstract— The aim of this study was to assess the capability of MRI to characterize systolic and diastolic function in normal and chronically failing mouse hearts in vivo at rest and during inotropic stimulation. Applying an ECG-gated FLASH-cine sequence, MRI at 7 T was performed at rest and after administration of 1.5 &mgr;g/g IP dobutamine. There was a significant increase of heart rate, cardiac output, and ejection fraction and significant decrease of end-diastolic and end-systolic left ventricular (LV) volumes (P <0.01 each) in normal mice during inotropic stimulation. In mice with heart failure due to chronic myocardial infarction (MI), MRI at rest revealed gross LV dilatation. There was a significant decrease of LV ejection fraction in infarcted mice (29%) versus sham mice (58%). Mice with MI showed a significantly reduced maximum LV ejection rate (P <0.001) and LV filling rate (P <0.01) and no increase of LV dynamics during dobutamine action, indicating loss of contractile and relaxation reserve. In 4-month-old transgenic mice with cardiospecific overexpression of the &bgr;1-adrenergic receptor, which at this early stage do not show abnormalities of resting cardiac function, LV filling rate failed to increase after dobutamine stress (transgenic, 0.19±0.03 &mgr;L/ms; wild type, 0.36±0.01 &mgr;L/ms;P <0.01). Thus, MRI unmasked diastolic dysfunction during dobutamine stress. Dobutamine-stress MRI allows noninvasive assessment of systolic and diastolic components of heart failure. This study shows that MRI can demonstrate loss of inotropic and lusitropic response in mice with MI and can unmask diastolic dysfunction as an early sign of cardiac dysfunction in a transgenic mouse model of heart failure.

Real-time optical recording of β1-adrenergic receptor activation reveals supersensitivity of the Arg389 variant to carvedilol

Antagonists of beta-adrenergic receptors (beta-ARs) have become a main therapeutic regimen for the treatment of heart failure even though the mechanisms of their beneficial effects are still poorly understood. Here, we used fluorescent resonance energy transfer-based (FRET-based) approaches to directly monitor activation of the beta(1)-AR and downstream signaling. While the commonly used beta-AR antagonists metoprolol, bisoprolol, and carvedilol displayed varying degrees of inverse agonism on the Gly389 variant of the receptor (i.e., actively switching off the beta(1)-AR), surprisingly, only carvedilol showed very specific and marked inverse agonist effects on the more frequent Arg389 variant. These specific effects of carvedilol on the Arg389 variant of the beta(1)-AR were also seen for control of beating frequency in rat cardiac myocytes expressing the 2 receptor variants. This FRET sensor permitted direct observation of activation of the beta(1)-AR in living cells in real time. It revealed that beta(1)-AR variants dramatically differ in their responses to diverse beta blockers, with possible consequences for their clinical use.