Britta Stapel

Continuous Glycoprotein-130–Mediated Signal Transducer and Activator of Transcription-3 Activation Promotes Inflammation, Left Ventricular Rupture, and Adverse Outcome in Subacute Myocardial Infarction

Background— In patients with myocardial infarction, high serum levels of interleukin-6 cytokines predict a poor outcome. The common receptor of interleukin-6 cytokines, glycoprotein-130 (gp130), signals via janus kinase/signal transducer and activator of transcription (STAT), cytoplasmic protein tyrosine phosphatase/extracellular signal-regulated kinase, and phosphoinositide-3-kinase/Akt pathways, and the regulation of these pathways depends at least in part on the gp130 tyrosine-757 residue. By analyzing cardiomyocyte-specific gp130Y757F mutant mice, we investigated the effect of disturbed gp130 signaling after myocardial infarction. Methods and Results— The cardiomyocyte-restricted &agr;-myosin heavy chain-Cre-recombinase-loxP system was used to generate mice with gp130Y757F mutant cardiomyocytes (&agr;MHC-Cretg/−;gp130fl/Y757F [Y757F]); all other cells carried at least 1 functional gp130 gene, ensuring normal gp130 signaling. Y757F mice displayed normal cardiac function and morphology at 3 months of age comparable to their nonmutant littermates. In response to myocardial infarction, Y757F mice displayed higher mortality associated with increased left ventricular rupture rate, sustained cardiac inflammation, and heart failure. These adverse effects were associated with prolonged and enhanced STAT3 activation and increased expression of interleukin-6 and of the complement-activating mannose-binding lectin C. Pharmacological inhibition of the complement system by cobra venom factor attenuated inflammation, prevented left ventricular rupture, and improved cardiac function in Y757F mice. Stronger effects were observed with a genetic reduction of STAT3 (STAT3flox/+) restricted to cardiomyocytes in Y757F mice, which prevented extensive upregulation of interleukin-6, complement activation, and sustained inflammation and lowered left ventricular rupture rate, heart failure, and mortality in subacute myocardial infarction. Conclusion— Impaired downregulation of gp130-mediated STAT3 activation in subacute infarction promotes cardiac inflammation, adverse remodeling, and heart failure, suggesting a potential causative role of high interleukin-6 serum levels after myocardial infarction.

Signal transducer and activator of transcription 3-mediated regulation of miR-199a-5p links cardiomyocyte and endothelial cell function in the heart: a key role for ubiquitin-conjugating enzymes.

AIMS Mice with a cardiomyocyte (CM)-restricted knockout of signal transducer and activator of transcription 3 (STAT3-KO) develop spontaneous heart failure. We investigated the impact of STAT3-mediated regulation of microRNAs for pathophysiological alterations in the heart. METHODS AND RESULTS MicroRNAchip and qRT-PCR analysis revealed elevated cardiac expression of miR-199a in STAT3-KO mice. Lentiviral shRNA-mediated STAT3-knock-down in neonatal rat CMs markedly increased miR-199a promoter activity and miR-199a levels indicative of a suppressive effect of STAT3 on miR-199a transcription. Up-regulated miR-199a in CM by pre-miR-199a transfection (pre-miR-199a-CM) reduced expression of components of the ubiquitin-proteasome system (UPS), i.e. the ubiquitin-conjugating enzymes Ube2g1 (mRNA and protein) and Ube2i (protein). Pre-miR-199a-CM or CM with siRNA-mediated down-regulation of Ube2i and Ube2g1 (siRNA-Ube2i/2g1-CM) displayed massive down-regulation of α- and β-myosin heavy chain expression associated with disrupted sarcomere structures. In addition, protein arginine methyltransferase I (PRMT-I) expression and asymmetric dimethylarginine (ADMA) synthesis were increased in pre-miR-199a-CM or in siRNA-Ube2i/2g1-CM. Increased ADMA in cell culture supernatant (SN) from pre-miR-199a-CM or siRNA-Ube2i/2g1-CM lowered nitric oxide (NO) bioavailability of rat cardiac endothelial cells while lowering ADMA concentration in CM SNs by the PRMT inhibitor arginine methyltransferase inhibitor 1 (AMI-1) (100 µM) improved NO bioavailability. In STAT3-KO hearts Ube2i and Ube2g1 expression were markedly reduced. Human terminal failing hearts harbouring low STAT3 protein levels displayed increased miR-199a levels and decreased Ube2g1 expression. CONCLUSION This study identifies a novel pathophysiological circuit in the heart between reduced STAT3 protein levels, increased miR-199a expression, and subsequent impairment of the UPS that disrupts CM sarcomere structure and impairs via the release of ADMA endothelial cell function.

STAT3 and cardiac remodeling

Multiple in vitro and in vivo studies showed that the signal transducer and activator of transcription 3 (STAT3) protein is involved in cardiomyocyte protection and hypertrophy and via paracrine pathways impacts on the non-myocyte compartment, i.e., the vasculature and the extracellular matrix. In this regard, STAT3 interacts with a broad range of cellular and molecular mechanisms that direct remodeling processes in cardiac physiology (exercise, pregnancy) and pathophysiology (pressure overload, ischemia/reperfusion, myocardial infarction, and cardiotoxic agents). STAT3 is constitutively activated by a multitude of factors including cytokines, growth factors, neurohormones, mechanical load, and ischemia. It acts as a signaling molecule, a transcription factor and according to latest observations as a mitochondrial protein involved in energy production. In this review, we provide an overview on STAT3 signaling and summarize the current understanding of the role of STAT3 for different aspects of cardiac remodeling obtained from numerous experimental and clinical studies. Finally, we highlight and critically discuss STAT3 signaling as a possible target for future therapeutic approaches in the setting of cardiac remodeling.

Erythropoietin Preserves the Endothelial Differentiation Capacity of Cardiac Progenitor Cells and Reduces Heart Failure during Anticancer Therapies

Anticancer therapies, such as targeting of STAT3 or the use of anthracyclins (doxorubicin), can induce cardiomyopathy. In mice prone to developing heart failure as a result of reduced cardiac STAT3 expression (cardiomyocyte-restricted deficiency of STAT3) or treatment with doxorubicin, we observed impaired endothelial differentiation capacity of Sca-1(+) cardiac progenitor cells (CPCs) in conjunction with attenuated CCL2/CCR2 activation. Mice in both models also displayed reduced erythropoietin (EPO) levels in the cardiac microenvironment. EPO binds to CPCs and seems to be responsible for maintaining an active CCL2/CCR2 system. Supplementation with the EPO derivative CERA in a hematocrit-inactive low dose was sufficient to upregulate CCL2, restore endothelial differentiation of CPCs, and preserve the cardiac microvasculature and cardiac function in both mouse models. Thus, low-dose EPO treatment could potentially be exploited as a therapeutic strategy to reduce the risk of heart failure in certain treatment regimens.

STAT3, a key regulator of cell-to-cell communication in the heart

The signal transducer and activator of transcription 3 (STAT3) is fundamental for physiological homeostasis and stress-induced remodelling of the heart as deregulated STAT3 circuits are sufficient to induce dilated and peripartum cardiomyopathy and adverse remodelling after myocardial infarction. STAT3 activity depends on multiple post-translational modifications (phosphorylation, acetylation, and dimerization). It is regulated by multiple receptor systems, which are coupled to positive and negative feedback loops to ensure physiological and beneficial action. Its intracellular functions are diverse as it acts as a signalling protein, a transcription factor but also participates in mitochondria energy production and protection. STAT3 modulates proliferation, differentiation, survival, oxidative stress, and/or metabolism in cardiomyocytes, fibroblasts, endothelial cells, progenitor cells, and various inflammatory cells. By regulating the secretome of these cardiac cells, STAT3 influences a broad range of intercellular communication systems. It thereby impacts on the communication between cardiomyocytes, the plasticity of the cardiac microenvironment, the vasculature, the extracellular matrix, and the inflammation in response to physiological and pathophysiological stress. Here, we sum up current knowledge on STAT3-mediated intra- and intercellular communication within the heterogeneous cellular network of the myocardium to co-ordinate complex biological processes and discuss STAT3-dependent targets as novel therapeutic concepts to treat various forms of heart disease.

Opposing roles of Akt and STAT3 in the protection of the maternal heart from peripartum stress

BACKGROUND Peripartum cardiomyopathy (PPCM) is a pregnancy-associated cardiomyopathy in previously healthy women. Mice with a cardiomyocyte-restricted deletion of signal transducer and activator of transcription-3 (STAT3, CKO) develop PPCM. PI3K-Akt signalling is thought to promote cardiac hypertrophy and protection during pregnancy. We evaluated the role of activated Akt signalling in the maternal heart postpartum. METHODS AND RESULTS CKO mice were bred to mice harbouring an Akt transgene, specifically expressed in cardiomyocytes (CAkt(tg)) generating CKO; CAkt(tg), CAkt(tg), CKO, and wild-type sibling mice. CAkt(tg) and CKO;CAkt(tg) female mice developed PPCM with systolic dysfunction. Both genotypes displayed cardiac hypertrophy and lower capillary density, showed increased phosphorylation of p66 Src homology 2 domain containing protein and FoxO3A, and reduced expression of manganese superoxide dismutase as well as increased cathepsin D activity and increased miR-146a levels [indicative for generation of the anti-angiogenic 16 kDa prolactin (PRL)]. Cardiac inflammation and fibrosis was accelerated in CKO;CAkt(tg) and associated with high postpartum mortality. The PRL blocker, bromocriptine (BR), prevented heart failure and the decrease in capillary density in CKO;CAkt(tg) and CAkt(tg) mice. BR attenuated high mortality, up-regulation of CCL2, and cardiac inflammation as well as fibrosis in CKO;CAkt(tg). PRL infusion induced cardiac inflammation in CKO;CAkt(tg) independent of pregnancy. In neonatal rat cardiomyocytes, PRL and interferon γ (IFNγ) induced the expression of CCL2 via activation of Akt. CONCLUSION Postpartum Akt activation is detrimental for the peripartum heart as it lowers anti-oxidative defence and in combination with low STAT3 conditions, accelerate cardiac inflammation and fibrosis. PRL and its cleaved 16 kDa form are central for Akt-induced PPCM as indicated by the protection from the disease by PRL blockade.

Small molecule-mediated refolding and activation of myosin motor function

The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an allosteric pocket in the myosin motor domain. EMD 57033-binding protects myosin against heat stress and thermal denaturation. In the presence of EMD 57033, ATP hydrolysis, coupling between actin and nucleotide binding sites, and actin affinity in the presence of ATP are increased more than 10-fold. Addition of EMD 57033 to heat-inactivated β-cardiac myosin is followed by refolding and reactivation of ATPase and motile activities. In heat-stressed cardiomyocytes expression of the stress-marker atrial natriuretic peptide is suppressed by EMD 57033. Thus, EMD 57033 displays a much wider spectrum of activities than those previously associated with small, drug-like compounds. Allosteric effectors that mediate refolding and enhance enzymatic function have the potential to improve the treatment of heart failure, myopathies, and protein misfolding diseases. DOI: http://dx.doi.org/10.7554/eLife.01603.001

Low STAT3 expression sensitizes to toxic effects of β-adrenergic receptor stimulation in peripartum cardiomyopathy

Abstract Aims The benefit of the β1-adrenergic receptor (β1-AR) agonist dobutamine for treatment of acute heart failure in peripartum cardiomyopathy (PPCM) is controversial. Cardiac STAT3 expression is reduced in PPCM patients. Mice carrying a cardiomyocyte-restricted deletion of STAT3 (CKO) develop PPCM. We hypothesized that STAT3-dependent signalling networks may influence the response to β-AR agonist treatment in PPCM patients and analysed this hypothesis in CKO mice. Methods and results Follow-up analyses in 27 patients with severe PPCM (left ventricular ejection fraction ≤25%) revealed that 19 of 20 patients not obtaining dobutamine improved cardiac function. All seven patients obtaining dobutamine received heart transplantation (n = 4) or left ventricular assist devices (n = 3). They displayed diminished myocardial triglyceride, pyruvate, and lactate content compared with non-failing controls. The β-AR agonist isoproterenol (Iso) induced heart failure with high mortality in postpartum female, in non-pregnant female and in male CKO, but not in wild-type mice. Iso induced heart failure and high mortality in CKO mice by impairing fatty acid and glucose uptake, thereby generating a metabolic deficit. The latter was governed by disturbed STAT3-dependent signalling networks, microRNA-199a-5p, microRNA-7a-5p, insulin/glucose transporter-4, and neuregulin/ErbB signalling. The resulting cardiac energy depletion and oxidative stress promoted dysfunction and cardiomyocyte loss inducing irreversible heart failure, which could be attenuated by the β1-AR blocker metoprolol or glucose-uptake-promoting drugs perhexiline and etomoxir. Conclusions Iso impairs glucose uptake, induces energy depletion, oxidative stress, dysfunction, and death in STAT3-deficient cardiomyocytes mainly via β1-AR stimulation. These cellular alterations may underlie the dobutamine-induced irreversible heart failure progression in PPCM patients who frequently display reduced cardiac STAT3 expression.

STAT3 regulation of and by microRNAs in development and disease

MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs acting at the post-transcriptional level where they promote mRNA degradation and block protein translation. Recent findings suggest that complex transcriptional and post-transcriptional circuits control miRNAs. STAT3 has emerged as an important regulator of their expression and biogenesis and, in turn, STAT3 signaling pathways are controlled by distinct miRNAs. We summarize the current knowledge on STAT3 mediated processing of individual miRNAs and contrariwise, the modulation of the STAT3 pathway by miRNAs in development and in pathophysiological conditions such as immune processes, infection, cancer, cardiovascular disease and pulmonary hypertension.

Serelaxin treatment promotes adaptive hypertrophy but does not prevent heart failure in experimental peripartum cardiomyopathy

Aims Peripartum cardiomyopathy (PPCM) is a systolic left ventricular dysfunction developing in the peripartum phase in previously healthy women. Relaxin-2 is a pregnancy hormone with potential beneficial effects in heart failure patients. We evaluated Relaxin-2 as a potential diagnostic marker and/or a therapeutic agent in PPCM. Methods and results In healthy peripartum women, serum Relaxin-2 levels (measured by ELISA in the second half of pregnancy) were elevated showing a decreasing trend in the first postpartum week and returned to non-pregnant levels thereafter. In PPCM patients diagnosed in the first postpartum week, serum Relaxin-2 levels were lower compared to healthy postpartum stage-matched controls. In PPCM patients diagnosed later (0.5–10 months postpartum) Relaxin-2 levels were in the range of non-pregnant controls and not different from healthy postpartum stage-matched controls. In mice, serum Relaxin-1 (functional equivalent of human Relaxin-2) was increased late in pregnancy and rapidly cleared in the first postpartum week. In mice with PPCM due to a cardiomyocyte-specific knockout of STAT3 (CKO) neither low nor high dose of recombinant Relaxin-2 (serelaxin, sRlx-LD: 30 µg/kg/day; sRlx-HD: 300 µg/kg/day) affected cardiac fibrosis, inflammation and heart failure but sRlx-HD increased capillary/cardiomyocyte ratio. sRlx-HD significantly increased heart/body weight ratio and cardiomyocyte cross-sectional area in postpartum CKO and wild-type mice without changing the foetal gene expression program (ANP or β-MHC). sRlx-HD augmented plasma Prolactin levels in both genotypes, which induced cardiac activation of STAT5. In vitro analyses showed that Prolactin induces cardiomyocyte hypertrophy via activation of STAT5. Conclusion Although Relaxin-2 levels seemed lower in PPCM patients diagnosed early postpartum, we observed a high pregnancy-related variance of serum Relaxin-2 levels peripartum making it unsuitable as a biomarker for this condition. Supplementation with sRlx may contribute to angiogenesis and compensatory hypertrophy in the diseased heart, but the effects are not sufficient to prevent heart failure in an experimental PPCM model.AIMS Peripartum cardiomyopathy (PPCM) is a systolic left ventricular dysfunction developing in the peripartum phase in previously healthy women. Relaxin-2 is a pregnancy hormone with potential beneficial effects in heart failure patients. We evaluated Relaxin-2 as a potential diagnostic marker and/or a therapeutic agent in PPCM. METHODS AND RESULTS In healthy peripartum women, serum Relaxin-2 levels (measured by ELISA in the second half of pregnancy) were elevated showing a decreasing trend in the first postpartum week and returned to non-pregnant levels thereafter. In PPCM patients diagnosed in the first postpartum week, serum Relaxin-2 levels were lower compared to healthy postpartum stage-matched controls. In PPCM patients diagnosed later (0.5-10 months postpartum) Relaxin-2 levels were in the range of non-pregnant controls and not different from healthy postpartum stage-matched controls. In mice, serum Relaxin-1 (functional equivalent of human Relaxin-2) was increased late in pregnancy and rapidly cleared in the first postpartum week. In mice with PPCM due to a cardiomyocyte-specific knockout of STAT3 (CKO) neither low nor high dose of recombinant Relaxin-2 (serelaxin, sRlx-LD: 30 µg/kg/day; sRlx-HD: 300 µg/kg/day) affected cardiac fibrosis, inflammation and heart failure but sRlx-HD increased capillary/cardiomyocyte ratio. sRlx-HD significantly increased heart/body weight ratio and cardiomyocyte cross-sectional area in postpartum CKO and wild-type mice without changing the foetal gene expression program (ANP or β-MHC). sRlx-HD augmented plasma Prolactin levels in both genotypes, which induced cardiac activation of STAT5. In vitro analyses showed that Prolactin induces cardiomyocyte hypertrophy via activation of STAT5. CONCLUSION Although Relaxin-2 levels seemed lower in PPCM patients diagnosed early postpartum, we observed a high pregnancy-related variance of serum Relaxin-2 levels peripartum making it unsuitable as a biomarker for this condition. Supplementation with sRlx may contribute to angiogenesis and compensatory hypertrophy in the diseased heart, but the effects are not sufficient to prevent heart failure in an experimental PPCM model.