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1.
Circ Res ; 125(4): 431-448, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31284834

RESUMO

RATIONALE: Gene expression profiles have been mainly determined by analysis of transcript abundance. However, these analyses cannot capture posttranscriptional gene expression control at the level of translation, which is a key step in the regulation of gene expression, as evidenced by the fact that transcript levels often poorly correlate with protein levels. Furthermore, genome-wide transcript profiling of distinct cell types is challenging due to the fact that lysates from tissues always represent a mixture of cells. OBJECTIVES: This study aimed to develop a new experimental method that overcomes both limitations and to apply this method to perform a genome-wide analysis of gene expression on the translational level in response to pressure overload. METHODS AND RESULTS: By combining ribosome profiling (Ribo-seq) with a ribosome-tagging approach (Ribo-tag), it was possible to determine the translated transcriptome in specific cell types from the heart. After pressure overload, we monitored the cardiac myocyte translatome by purifying tagged cardiac myocyte ribosomes from cardiac lysates and subjecting the ribosome-protected mRNA fragments to deep sequencing. We identified subsets of mRNAs that are regulated at the translational level and found that translational control determines early changes in gene expression in response to cardiac stress in cardiac myocytes. Translationally controlled transcripts are associated with specific biological processes related to translation, protein quality control, and metabolism. Mechanistically, Ribo-seq allowed for the identification of upstream open reading frames in transcripts, which we predict to be important regulators of translation. CONCLUSIONS: This method has the potential to (1) provide a new tool for studying cell-specific gene expression at the level of translation in tissues, (2) reveal new therapeutic targets to prevent cellular remodeling, and (3) trigger follow-up studies that address both, the molecular mechanisms involved in the posttranscriptional control of gene expression in cardiac cells, and the protective functions of proteins expressed in response to cellular stress.

2.
Circulation ; 140(7): 580-594, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31195810

RESUMO

BACKGROUND: Worldwide, diabetes mellitus and heart failure represent frequent comorbidities with high socioeconomic impact and steadily growing incidence, calling for a better understanding of how diabetic metabolism promotes cardiac dysfunction. Paradoxically, some glucose-lowering drugs have been shown to worsen heart failure, raising the question of how glucose mediates protective versus detrimental cardiac signaling. Here, we identified a histone deacetylase 4 (HDAC4) subdomain as a molecular checkpoint of adaptive and maladaptive signaling in the diabetic heart. METHODS: A conditional HDAC4 allele was used to delete HDAC4 specifically in cardiomyocytes (HDAC4-knockout). Mice were subjected to diabetes mellitus either by streptozotocin injections (type 1 diabetes mellitus model) or by crossing into mice carrying a leptin receptor mutation (db/db; type 2 diabetes mellitus model) and monitored for remodeling and cardiac function. Effects of glucose and the posttranslational modification by ß-linked N-acetylglucosamine (O-GlcNAc) on HDAC4 were investigated in vivo and in vitro by biochemical and cellular assays. RESULTS: We show that the cardio-protective N-terminal proteolytic fragment of HDAC4 is enhanced in vivo in patients with diabetes mellitus and mouse models, as well as in vitro under high-glucose and high-O-GlcNAc conditions. HDAC4-knockout mice develop heart failure in models of type 1 and type 2 diabetes mellitus, whereas wild-type mice do not develop clear signs of heart failure, indicating that HDAC4 protects the diabetic heart. Reexpression of the N-terminal fragment of HDAC4 prevents HDAC4-dependent diabetic cardiomyopathy. Mechanistically, the posttranslational modification of HDAC4 at serine (Ser)-642 by O-GlcNAcylation is an essential step for production of the N-terminal fragment of HDAC4, which was attenuated by Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation at Ser-632. Preventing O-GlcNAcylation at Ser-642 not only entirely precluded production of the N-terminal fragment of HDAC4 but also promoted Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation at Ser-632, pointing to a mutual posttranslational modification cross talk of (cardio-detrimental) phosphorylation at Ser-632 and (cardio-protective) O-GlcNAcylation at Ser-642. CONCLUSIONS: In this study, we found that O-GlcNAcylation of HDAC4 at Ser-642 is cardio-protective in diabetes mellitus and counteracts pathological Ca2+/calmodulin-dependent protein kinase II signaling. We introduce a molecular model explaining how diabetic metabolism possesses important cardio-protective features besides its known detrimental effects. A deeper understanding of the here-described posttranslational modification cross talk may lay the groundwork for the development of specific therapeutic concepts to treat heart failure in the context of diabetes mellitus.

3.
Eur J Heart Fail ; 21(5): 553-576, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30989768

RESUMO

Cardiomyopathies are a heterogeneous group of heart muscle diseases and an important cause of heart failure (HF). Current knowledge on incidence, pathophysiology and natural history of HF in cardiomyopathies is limited, and distinct features of their therapeutic responses have not been systematically addressed. Therefore, this position paper focuses on epidemiology, pathophysiology, natural history and latest developments in treatment of HF in patients with dilated (DCM), hypertrophic (HCM) and restrictive (RCM) cardiomyopathies. In DCM, HF with reduced ejection fraction (HFrEF) has high incidence and prevalence and represents the most frequent cause of death, despite improvements in treatment. In addition, advanced HF in DCM is one of the leading indications for heart transplantation. In HCM, HF with preserved ejection (HFpEF) affects most patients with obstructive, and ∼10% of patients with non-obstructive HCM. A timely treatment is important, since development of advanced HF, although rare in HCM, portends a poor prognosis. In RCM, HFpEF is common, while HFrEF occurs later and more frequently in amyloidosis or iron overload/haemochromatosis. Irrespective of RCM aetiology, HF is a harbinger of a poor outcome. Recent advances in our understanding of the mechanisms underlying the development of HF in cardiomyopathies have significant implications for therapeutic decision-making. In addition, new aetiology-specific treatment options (e.g. enzyme replacement therapy, transthyretin stabilizers, immunoadsorption, immunotherapy, etc.) have shown a potential to improve outcomes. Still, causative therapies of many cardiomyopathies are lacking, highlighting the need for the development of effective strategies to prevent and treat HF in cardiomyopathies.

4.
ESC Heart Fail ; 6(4): 711-722, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31025825

RESUMO

AIMS: Myocardial infarction (MI) and heart failure (HF) are risk factors for the development of depression, additionally worsening the quality of life and patient outcome. How HF causes depression and how depression promotes HF remain mechanistically unclear, which is at least partly caused by the difficulty of in vivo modelling of psychosomatic co-morbidity. We aimed to study the potential sequence of events with respect to different depression aspects upon HF. METHODS AND RESULTS: Male C57BL6 mice underwent MI, followed by behavioural and echocardiographic characterization. Motility, exploration, and anxiety-like behaviour were unaffected in mice after MI. We did not observe increased depressive-like behaviour in the sucrose preference, tail suspension, or Porsolt forced swim test. Mice did not display signs of learned helplessness (LH) when compared to sham. Accordingly, cluster analysis revealed only a slightly higher quota of LH in HF (38%) vs. sham mice (32%). But strikingly, three-group cluster analysis revealed an additional intermediate subpopulation at risk for LH after HF (29%). Interestingly, this population featured elevated cardiac expression of nr4a1. CONCLUSIONS: The LH paradigm uncovered a subtle predisposition to depressive-like behaviour after MI, whereas testing for anhedonia and despair was insufficient to show a behavioural shift in mice. Therefore, we suggest an accumulating risk profile and a multiple-hits hypothesis regarding the pathogenesis of co-morbid depression after MI. Symptoms of LH may present a marker of subclinical depression after MI, the impact of which remains to be investigated. The proposed sequence of behavioural testing enables the mechanistic dissection of cardio-psychogenic signalling in the future.

5.
Eur Heart J ; 40(26): 2155-2163, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30957868

RESUMO

Randomized clinical trials initially used heart failure (HF) patients with low left ventricular ejection fraction (LVEF) to select study populations with high risk to enhance statistical power. However, this use of LVEF in clinical trials has led to oversimplification of the scientific view of a complex syndrome. Descriptive terms such as 'HFrEF' (HF with reduced LVEF), 'HFpEF' (HF with preserved LVEF), and more recently 'HFmrEF' (HF with mid-range LVEF), assigned on arbitrary LVEF cut-off points, have gradually arisen as separate diseases, implying distinct pathophysiologies. In this article, based on pathophysiological reasoning, we challenge the paradigm of classifying HF according to LVEF. Instead, we propose that HF is a heterogeneous syndrome in which disease progression is associated with a dynamic evolution of functional and structural changes leading to unique disease trajectories creating a spectrum of phenotypes with overlapping and distinct characteristics. Moreover, we argue that by recognizing the spectral nature of the disease a novel stratification will arise from new technologies and scientific insights that will shape the design of future trials based on deeper understanding beyond the LVEF construct alone.

6.
J Physiol ; 2019 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-30770570

RESUMO

KEY POINTS: Mitochondrial Ca2+ uptake stimulates the Krebs cycle to regenerate the reduced forms of pyridine nucleotides (NADH, NADPH and FADH2 ) required for ATP production and reactive oxygen species (ROS) elimination. Ca2+ /calmodulin-dependent protein kinase II (CaMKII) has been proposed to regulate mitochondrial Ca2+ uptake via mitochondrial Ca2+ uniporter phosphorylation. We used two mouse models with either global deletion of CaMKIIδ (CaMKIIδ knockout) or cardiomyocyte-specific deletion of CaMKIIδ and γ (CaMKIIδ/γ double knockout) to interrogate whether CaMKII controls mitochondrial Ca2+ uptake in isolated mitochondria and during ß-adrenergic stimulation in cardiac myocytes. CaMKIIδ/γ did not control Ca2+ uptake, respiration or ROS emission in isolated cardiac mitochondria, nor in isolated cardiac myocytes, during ß-adrenergic stimulation and pacing. The results of the present study do not support a relevant role of CaMKII for mitochondrial Ca2+ uptake in cardiac myocytes under physiological conditions. ABSTRACT: Mitochondria are the main source of ATP and reactive oxygen species (ROS) in cardiac myocytes. Furthermore, activation of the mitochondrial permeability transition pore (mPTP) induces programmed cell death. These processes are essentially controlled by Ca2+ , which is taken up into mitochondria via the mitochondrial Ca2+ uniporter (MCU). It was recently proposed that Ca2+ /calmodulin-dependent protein kinase II (CaMKII) regulates Ca2+ uptake by interacting with the MCU, thereby affecting mPTP activation and programmed cell death. In the present study, we investigated the role of CaMKII under physiological conditions in which mitochondrial Ca2+ uptake matches energy supply to the demand of cardiac myocytes. Accordingly, we measured mitochondrial Ca2+ uptake in isolated mitochondria and cardiac myocytes harvested from cardiomyocyte-specific CaMKII δ and γ double knockout (KO) (CaMKIIδ/γ DKO) and global CaMKIIδ KO mice. To simulate a physiological workload increase, cardiac myocytes were subjected to ß-adrenergic stimulation (by isoproterenol superfusion) and an increase in stimulation frequency (from 0.5 to 5 Hz). No differences in mitochondrial Ca2+ accumulation were detected in isolated mitochondria or cardiac myocytes from both CaMKII KO models compared to wild-type littermates. Mitochondrial redox state and ROS production were unchanged in CaMKIIδ/γ DKO, whereas we observed a mild oxidation of mitochondrial redox state and an increase in H2 O2 emission from CaMKIIδ KO cardiac myocytes exposed to an increase in workload. In conclusion, the results obtained in the present study do not support the regulation of mitochondrial Ca2+ uptake via the MCU or mPTP activation by CaMKII in cardiac myocytes under physiological conditions.

7.
Cardiovasc Res ; 2018 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-30418544

RESUMO

Aims: Heart failure is characterized by structural and metabolic cardiac remodelling. The aim of the present study is to expand our understanding of the complex metabolic alterations in the transition from pathological hypertrophy to heart failure and exploit the results from a translational perspective. Methods and results: Mice were subjected to transverse aortic constriction (TAC) or sham surgery and sacrificed 2, 4, or 6 weeks after the procedure. Samples from plasma, liver, skeletal muscle, and heart were collected and analysed using metabolomics. Cardiac samples were also analysed by transcriptional profiling. Progressive alterations of key cardiac metabolic pathways and gene expression patterns indicated impaired mitochondrial function and a metabolic switch during transition to heart failure. Similar to the heart, liver and skeletal muscle revealed significant metabolic alterations such as depletion of essential fatty acids and glycerolipids in late stages of heart failure. Circulating metabolites, particularly fatty acids, reflected cardiac metabolic defects and deteriorating heart function. For example, inverse correlation was found between plasma and the heart levels of triacylglycerol (C18:1, C18:2, C18:3), and sphingomyelin (d18:1, C23:0) already at an early stage of heart failure. Interestingly, combining metabolic and transcriptional data from cardiac tissue revealed that decreased carnitine shuttling and transportation preceded mitochondrial dysfunction. We, thus, studied the therapeutic potential of OCTN2 (Organic Cation/Carnitine Transporter 2), an important factor for carnitine transportation. Cardiac overexpression of OCTN2 using an adeno-associated viral (AAV) vector significantly improved ejection fraction and reduced interstitial fibrosis in mice subjected to TAC. Conclusions: Comprehensive plasma and tissue profiling reveals systemic metabolic alterations in heart failure, which can be used for identification of novel biomarkers and potential therapeutic targets.

8.
Artigo em Inglês | MEDLINE | ID: mdl-30378296

RESUMO

BACKGROUND: Stroke can lead to cardiac dysfunction in patients, but the mechanisms underlying the interaction between the injured brain and the heart are poorly understood. The objective of the study is to investigate the effects of experimental murine stroke on cardiac function and molecular signalling in the heart. METHODS AND RESULTS: Mice were subjected to filament-induced left middle cerebral artery occlusion for 30 or 60 min or sham surgery and underwent repetitive micro-echocardiography. Left ventricular contractility was reduced early (24-72 h) but not late (2 months) after brain ischaemia. Cardiac dysfunction was accompanied by a release of high-sensitive cardiac troponin (hsTNT (ng/ml): d1: 7.0 ± 1.0 vs. 25.0 ± 3.2*; d3: 7.3 ± 1.1 vs. 52.2 ± 16.7*; d14: 5.7 ± 0.8 vs. 5.2 ± 0.3; sham vs. 60 min. MCAO; mean ± SEM; *p < 0.05); reduced heart weight (heart weight/tibia length ratio: d1: 6.9 ± 0.2 vs. 6.4 ± 0.1*; d3: 6.7 ± 0.2 vs. 5.8 ± 0.1*; d14: 6.7 ± 0.2 vs. 6.4 ± 03; sham vs. 60 min. MCAO; mean ± SEM; *p < 0.05); resulting from cardiomyocyte atrophy (cardiomyocyte size: d1: 12.8% ± 0.002**; d3: 13.5% ± 0.002**; 14d: 6.3% ± 0.003*; 60 min. MCAO vs. sham; mean ± SEM; **p < 0.01; *p < 0.05), accompanied by increased atrogin-1 and the E3 ubiquitin ligase murf-1. Net norepinephrine but not synthesis was increased, suggesting a reduced norepinephrine release or an increase of norepinephrine re-uptake, resulting in a functional denervation. Transcriptome analysis in cardiac tissue identified the transcription factor peroxisome proliferator-activated receptor gamma as a potential mediator of stroke-induced transcriptional dysregulation involved in cardiac atrophy. CONCLUSIONS: Stroke induces a complex molecular response in the heart muscle with immediate but transient cardiac atrophy and dysfunction.

10.
Basic Res Cardiol ; 113(4): 29, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29905892

RESUMO

In the cardiomyocyte, CaMKII has been identified as a nodal influencer of excitation-contraction and also excitation-transcription coupling. Its activity can be regulated in response to changes in intracellular calcium content as well as after several post-translational modifications. Some of the effects mediated by CaMKII may be considered adaptive, while effects of sustained CaMKII activity may turn into the opposite and are detrimental to cardiac integrity and function. As such, CaMKII has long been noted as a promising target for pharmacological inhibition, but the ubiquitous nature of CaMKII has made it difficult to target CaMKII specifically where it is detrimental. In this review, we provide a brief overview of the physiological and pathophysiological properties of CaMKII signaling, but we focus on the physiological and adaptive functions of CaMKII. Furthermore, special consideration is given to the emerging role of CaMKII as a mediator of inflammatory processes in the heart.

11.
EMBO Mol Med ; 10(7)2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29907596

RESUMO

The myocyte enhancer factor 2 (MEF2) regulates transcription in cardiac myocytes and adverse remodeling of adult hearts. Activators of G protein-coupled receptors (GPCRs) have been reported to activate MEF2, but a comprehensive analysis of GPCR activators that regulate MEF2 has to our knowledge not been performed. Here, we tested several GPCR agonists regarding their ability to activate a MEF2 reporter in neonatal rat ventricular myocytes. The inflammatory mediator prostaglandin E2 (PGE2) strongly activated MEF2. Using pharmacological and protein-based inhibitors, we demonstrated that PGE2 regulates MEF2 via the EP3 receptor, the ßγ subunit of Gi/o protein and two concomitantly activated downstream pathways. The first consists of Tiam1, Rac1, and its effector p21-activated kinase 2, the second of protein kinase D. Both pathways converge on and inactivate histone deacetylase 5 (HDAC5) and thereby de-repress MEF2. In vivo, endotoxemia in MEF2-reporter mice induced upregulation of PGE2 and MEF2 activation. Our findings provide an unexpected new link between inflammation and cardiac remodeling by de-repression of MEF2 through HDAC5 inactivation, which has potential implications for new strategies to treat inflammatory cardiomyopathies.

12.
Circulation ; 138(13): 1330-1342, 2018 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-29650543

RESUMO

BACKGROUND: Mutations in RBM20 (RNA-binding motif protein 20) cause a clinically aggressive form of dilated cardiomyopathy, with an increased risk of malignant ventricular arrhythmias. RBM20 is a splicing factor that targets multiple pivotal cardiac genes, such as Titin (TTN) and CAMK2D (calcium/calmodulin-dependent kinase II delta). Aberrant TTN splicing is thought to be the main determinant of RBM20-induced dilated cardiomyopathy, but is not likely to explain the increased risk of arrhythmias. Here, we investigated the extent to which RBM20 mutation carriers have an increased risk of arrhythmias and explore the underlying molecular mechanism. METHODS: We compared clinical characteristics of RBM20 and TTN mutation carriers and used our previously generated Rbm20 knockout (KO) mice to investigate downstream effects of Rbm20-dependent splicing. Cellular electrophysiology and Ca2+ measurements were performed on isolated cardiomyocytes from Rbm20 KO mice to determine the intracellular consequences of reduced Rbm20 levels. RESULTS: Sustained ventricular arrhythmias were more frequent in human RBM20 mutation carriers than in TTN mutation carriers (44% versus 5%, respectively, P=0.006). Splicing events that affected Ca2+- and ion-handling genes were enriched in Rbm20 KO mice, most notably in the genes CamkIIδ and RyR2. Aberrant splicing of CamkIIδ in Rbm20 KO mice resulted in a remarkable shift of CamkIIδ toward the δ-A isoform that is known to activate the L-type Ca2+ current ( ICa,L). In line with this, we found an increased ICa,L, intracellular Ca2+ overload and increased sarcoplasmic reticulum Ca2+ content in Rbm20 KO myocytes. In addition, not only complete loss of Rbm20, but also heterozygous loss of Rbm20 increased spontaneous sarcoplasmic reticulum Ca2+ releases, which could be attenuated by treatment with the ICa,L antagonist verapamil. CONCLUSIONS: We show that loss of Rbm20 disturbs Ca2+ handling and leads to more proarrhythmic Ca2+ releases from the sarcoplasmic reticulum. Patients that carry a pathogenic RBM20 mutation have more ventricular arrhythmias despite a similar left ventricular function, in comparison with patients with a TTN mutation. Our experimental data suggest that RBM20 mutation carriers may benefit from treatment with an ICa,L blocker to reduce their arrhythmia burden.

13.
J Cell Biol ; 217(4): 1249-1268, 2018 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-29472387

RESUMO

Schwann cells respond to cyclic adenosine monophosphate (cAMP) halting proliferation and expressing myelin proteins. Here we show that cAMP signaling induces the nuclear shuttling of the class IIa histone deacetylase (HDAC)-4 in these cells, where it binds to the promoter and blocks the expression of c-Jun, a negative regulator of myelination. To do it, HDAC4 does not interfere with the transcriptional activity of MEF2. Instead, by interacting with NCoR1, it recruits HDAC3 and deacetylates histone 3 in the promoter of c-Jun, blocking gene expression. Importantly, this is enough to up-regulate Krox20 and start Schwann cell differentiation program-inducing myelin gene expression. Using conditional knockout mice, we also show that HDAC4 together with HDAC5 redundantly contribute to activate the myelin transcriptional program and the development of myelin sheath in vivo. We propose a model in which cAMP signaling shuttles class IIa HDACs into the nucleus of Schwann cells to regulate the initial steps of myelination in the peripheral nervous system.

14.
Circulation ; 137(24): 2592-2608, 2018 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-29353241

RESUMO

BACKGROUND: Nutrients are transported through endothelial cells before being metabolized in muscle cells. However, little is known about the regulation of endothelial transport processes. Notch signaling is a critical regulator of metabolism and angiogenesis during development. Here, we studied how genetic and pharmacological manipulation of endothelial Notch signaling in adult mice affects endothelial fatty acid transport, cardiac angiogenesis, and heart function. METHODS: Endothelial-specific Notch inhibition was achieved by conditional genetic inactivation of Rbp-jκ in adult mice to analyze fatty acid metabolism and heart function. Wild-type mice were treated with neutralizing antibodies against the Notch ligand Delta-like 4. Fatty acid transport was studied in cultured endothelial cells and transgenic mice. RESULTS: Treatment of wild-type mice with Delta-like 4 neutralizing antibodies for 8 weeks impaired fractional shortening and ejection fraction in the majority of mice. Inhibition of Notch signaling specifically in the endothelium of adult mice by genetic ablation of Rbp-jκ caused heart hypertrophy and failure. Impaired heart function was preceded by alterations in fatty acid metabolism and an increase in cardiac blood vessel density. Endothelial Notch signaling controlled the expression of endothelial lipase, Angptl4, CD36, and Fabp4, which are all needed for fatty acid transport across the vessel wall. In endothelial-specific Rbp-jκ-mutant mice, lipase activity and transendothelial transport of long-chain fatty acids to muscle cells were impaired. In turn, lipids accumulated in the plasma and liver. The attenuated supply of cardiomyocytes with long-chain fatty acids was accompanied by higher glucose uptake, increased concentration of glycolysis intermediates, and mTOR-S6K signaling. Treatment with the mTOR inhibitor rapamycin or displacing glucose as cardiac substrate by feeding a ketogenic diet prolonged the survival of endothelial-specific Rbp-jκ-deficient mice. CONCLUSIONS: This study identifies Notch signaling as a novel regulator of fatty acid transport across the endothelium and as an essential repressor of angiogenesis in the adult heart. The data imply that the endothelium controls cardiomyocyte metabolism and function.

15.
Nat Med ; 24(1): 62-72, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29227474

RESUMO

The stress-responsive epigenetic repressor histone deacetylase 4 (HDAC4) regulates cardiac gene expression. Here we show that the levels of an N-terminal proteolytically derived fragment of HDAC4, termed HDAC4-NT, are lower in failing mouse hearts than in healthy control hearts. Virus-mediated transfer of the portion of the Hdac4 gene encoding HDAC4-NT into the mouse myocardium protected the heart from remodeling and failure; this was associated with decreased expression of Nr4a1, which encodes a nuclear orphan receptor, and decreased NR4A1-dependent activation of the hexosamine biosynthetic pathway (HBP). Conversely, exercise enhanced HDAC4-NT levels, and mice with a cardiomyocyte-specific deletion of Hdac4 show reduced exercise capacity, which was characterized by cardiac fatigue and increased expression of Nr4a1. Mechanistically, we found that NR4A1 negatively regulated contractile function in a manner that depended on the HBP and the calcium sensor STIM1. Our work describes a new regulatory axis in which epigenetic regulation of a metabolic pathway affects calcium handling. Activation of this axis during intermittent physiological stress promotes cardiac function, whereas its impairment in sustained pathological cardiac stress leads to heart failure.

16.
Contrast Media Mol Imaging ; 2017: 2617047, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29114173

RESUMO

We herein developed a micro-CT method using the innovative contrast agent ExiTron™ MyoC 8000 to longitudinally monitor cardiac processes in vivo in small animals. Experiments were performed on healthy mice and mice with myocardial infarction inflicted by ligation of the left anterior descending artery. Time-dependent signal enhancement in different tissues of healthy mice was measured and various contrast agent doses were investigated so as to determine the minimum required dose for imaging of the myocardium. Due to its ability to be taken up by healthy myocardium but not by infarct tissue, ExiTron MyoC 8000 enables detection of myocardial infarction even at a very low dose. The signal enhancement in the myocardium of infarcted mice after contrast agent injection was exploited for quantification of infarct size. The values of infarct size obtained from the imaging method were compared with those obtained from histology and showed a significant correlation (R2 = 0.98). Thus, the developed micro-CT method allows for monitoring of a variety of processes such as cardiac remodeling in longitudinal studies.


Assuntos
Meios de Contraste/farmacologia , Infarto do Miocárdio/diagnóstico por imagem , Microtomografia por Raio-X , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Camundongos
17.
Circ Res ; 121(8): 1000-1020, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28963192

RESUMO

Calcium (Ca2+) is a universal regulator of various cellular functions. In cardiomyocytes, Ca2+ is the central element of excitation-contraction coupling, but also impacts diverse signaling cascades and influences the regulation of gene expression, referred to as excitation-transcription coupling. Disturbances in cellular Ca2+-handling and alterations in Ca2+-dependent gene expression patterns are pivotal characteristics of failing cardiomyocytes, with several excitation-transcription coupling pathways shown to be critically involved in structural and functional remodeling processes. Thus, targeting Ca2+-dependent transcriptional pathways might offer broad therapeutic potential. In this article, we (1) review cytosolic and nuclear Ca2+ dynamics in cardiomyocytes with respect to their impact on Ca2+-dependent signaling, (2) give an overview on Ca2+-dependent transcriptional pathways in cardiomyocytes, and (3) discuss implications of excitation-transcription coupling in the diseased heart.


Assuntos
Sinalização do Cálcio , Cardiopatias/genética , Cardiopatias/metabolismo , Miócitos Cardíacos/metabolismo , Transcrição Genética , Animais , Sinalização do Cálcio/efeitos dos fármacos , Fármacos Cardiovasculares/uso terapêutico , Núcleo Celular/metabolismo , Citosol/metabolismo , Acoplamento Excitação-Contração , Regulação da Expressão Gênica , Cardiopatias/tratamento farmacológico , Cardiopatias/fisiopatologia , Humanos , Cinética , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
18.
PLoS One ; 12(7): e0182110, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28759639

RESUMO

The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3ß), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase γ (PI3Kγ). Moreover, inhibition of PI3Kγ as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3Kγ mediated Akt phosphorylation and subsequent inactivation of GSK3ß, resulting in enhanced NFAT activity.


Assuntos
Cardiomegalia/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Animais , Cardiomegalia/genética , Células Cultivadas , Quinase 2 de Receptor Acoplado a Proteína G/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Linfócitos T/imunologia
19.
Circulation ; 136(16): 1528-1544, 2017 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-28838933

RESUMO

BACKGROUND: Biochemical DNA modification resembles a crucial regulatory layer among genetic information, environmental factors, and the transcriptome. To identify epigenetic susceptibility regions and novel biomarkers linked to myocardial dysfunction and heart failure, we performed the first multi-omics study in myocardial tissue and blood of patients with dilated cardiomyopathy and controls. METHODS: Infinium human methylation 450 was used for high-density epigenome-wide mapping of DNA methylation in left-ventricular biopsies and whole peripheral blood of living probands. RNA deep sequencing was performed on the same samples in parallel. Whole-genome sequencing of all patients allowed exclusion of promiscuous genotype-induced methylation calls. RESULTS: In the screening stage, we detected 59 epigenetic loci that are significantly associated with dilated cardiomyopathy (false discovery corrected P≤0.05), with 3 of them reaching epigenome-wide significance at P≤5×10-8. Twenty-seven (46%) of these loci could be replicated in independent cohorts, underlining the role of epigenetic regulation of key cardiac transcription regulators. Using a staged multi-omics study design, we link a subset of 517 epigenetic loci with dilated cardiomyopathy and cardiac gene expression. Furthermore, we identified distinct epigenetic methylation patterns that are conserved across tissues, rendering these CpGs novel epigenetic biomarkers for heart failure. CONCLUSIONS: The present study provides to our knowledge the first epigenome-wide association study in living patients with heart failure using a multi-omics approach.


Assuntos
Cardiomiopatia Dilatada/genética , Metilação de DNA , Epigênese Genética , Epigenômica/métodos , Loci Gênicos , Insuficiência Cardíaca/genética , Ventrículos do Coração/química , Cardiomiopatia Dilatada/sangue , Cardiomiopatia Dilatada/diagnóstico , Estudos de Casos e Controles , Ilhas de CpG , Perfilação da Expressão Gênica , Marcadores Genéticos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Fenótipo , RNA Mensageiro/genética , Análise de Sequência de RNA
20.
Circ Heart Fail ; 10(5): e003840, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28487342

RESUMO

BACKGROUND: Considerable evidence suggests that calcium/calmodulin-dependent protein kinase II (CaMKII) overactivity plays a crucial role in the pathophysiology of heart failure (HF), a condition characterized by excessive ß-adrenoceptor (ß-AR) stimulation. Recent studies indicate a significant cross talk between ß-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental ß-AR signaling in HF. In this study, we investigated the effect of chronic ß-AR blocker treatment on CaMKII activity in human and experimental HF. METHODS AND RESULTS: Immunoblot analysis of myocardium from end-stage HF patients (n=12) and non-HF subjects undergoing cardiac surgery (n=12) treated with ß-AR blockers revealed no difference in CaMKII activity when compared with non-ß-AR blocker-treated patients. CaMKII activity was judged by analysis of CaMKII expression, autophosphorylation, and oxidation and by investigating the phosphorylation status of CaMKII downstream targets. To further evaluate these findings, CaMKIIδC transgenic mice were treated with the ß1-AR blocker metoprolol (270 mg/kg*d). Metoprolol significantly reduced transgene-associated mortality (n≥29; P<0.001), attenuated the development of cardiac hypertrophy (-14±6% heart weight/tibia length; P<0.05), and strongly reduced ventricular arrhythmias (-70±22% premature ventricular contractions; P<0.05). On a molecular level, metoprolol expectedly decreased protein kinase A-dependent phospholamban and ryanodine receptor 2 phosphorylation (-42±9% for P-phospholamban-S16 and -22±7% for P-ryanodine receptor 2-S2808; P<0.05). However, this was paralled neither by a reduction in CaMKII autophosphorylation, oxidation, and substrate binding nor a change in the phosphorylation of CaMKII downstream target proteins (n≥11). The lack of CaMKII modulation by ß-AR blocker treatment was confirmed in healthy wild-type mice receiving metoprolol. CONCLUSIONS: Chronic ß-AR blocker therapy in patients and in a mouse model of CaMKII-induced HF is not associated with a change in CaMKII activity. Thus, our data suggest that the molecular effects of ß-AR blockers are not based on a modulation of CaMKII. Directly targeting CaMKII may, therefore, further improve HF therapy in addition to ß-AR blockade.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Insuficiência Cardíaca/metabolismo , Metoprolol/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Modelos Animais de Doenças , Ecocardiografia , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/tratamento farmacológico , Humanos , Immunoblotting , Camundongos , Camundongos Transgênicos
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