Genomic Binding Patterns of Forkhead Box Protein O1 Reveal Its Unique Role in Cardiac Hypertrophy


<jats:sec> <jats:title>Background:</jats:title> <jats:p>Cardiac hypertrophic growth is mediated by robust changes in gene expression and changes that underlie the increase in cardiomyocyte size. The former is regulated by RNA polymerase II (pol II) de novo recruitment or loss; the latter involves incremental increases in the transcriptional elongation activity of pol II that is preassembled at the transcription start site. The differential regulation of these distinct processes by transcription factors remains unknown. Forkhead box protein O1 (FoxO1) is an insulin-sensitive transcription factor that is also regulated by hypertrophic stimuli in the heart. However, the scope of its gene regulation remains unexplored.</jats:p> </jats:sec> <jats:sec> <jats:title>Methods:</jats:title> <jats:p> To address this, we performed FoxO1 chromatin immunoprecipitation–deep sequencing in mouse hearts after 7 days of isoproterenol injections (3 mg·kg <jats:sup>−1</jats:sup> ·mg <jats:sup>−1</jats:sup> ), transverse aortic constriction, or vehicle injection/sham surgery. </jats:p> </jats:sec> <jats:sec> <jats:title>Results:</jats:title> <jats:p> Our data demonstrate increases in FoxO1 chromatin binding during cardiac hypertrophic growth, which positively correlate with extent of hypertrophy. To assess the role of FoxO1 on pol II dynamics and gene expression, the FoxO1 chromatin immunoprecipitation–deep sequencing results were aligned with those of pol II chromatin immunoprecipitation–deep sequencing across the chromosomal coordinates of sham- or transverse aortic constriction–operated mouse hearts. This uncovered that FoxO1 binds to the promoters of 60% of cardiac-expressed genes at baseline and 91% after transverse aortic constriction. FoxO1 binding is increased in genes regulated by pol II de novo recruitment, loss, or pause-release. In vitro, endothelin-1– and, in vivo, pressure overload–induced cardiomyocyte hypertrophic growth is prevented with FoxO1 knockdown or deletion, which was accompanied by reductions in inducible genes, including <jats:italic>Comtd1</jats:italic> in vitro and <jats:italic>Fstl1</jats:italic> and <jats:italic>Uck2</jats:italic> in vivo. </jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions:</jats:title> <jats:p>Together, our data suggest that FoxO1 may mediate cardiac hypertrophic growth via regulation of pol II de novo recruitment and pause-release; the latter represents the majority (59%) of FoxO1-bound, pol II–regulated genes after pressure overload. These findings demonstrate the breadth of transcriptional regulation by FoxO1 during cardiac hypertrophy, information that is essential for its therapeutic targeting.</jats:p> </jats:sec>






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Publication Info

Pfleger, Jessica, Ryan C Coleman, Jessica Ibetti, Rajika Roy, Ioannis D Kyriazis, Erhe Gao, Konstantinos Drosatos, Walter J Koch, et al. (2020). Genomic Binding Patterns of Forkhead Box Protein O1 Reveal Its Unique Role in Cardiac Hypertrophy. Circulation, 142(9). pp. 882–898. 10.1161/circulationaha.120.046356 Retrieved from

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Rajika Roy

Assistant Professor in Surgery

Walter J. Koch

Professor in Surgery

My research interests are centered on the molecular mechanisms involved in the regulation of signaling through cardiovascular adrenergic receptors (ARs) including the specific in vivo interactions between ARs and myocardial G protein-coupled receptor kinases (GRKs). This includes the role of ARs and GRKs in cardiovascular disease. The β-adrenergic receptor kinase (betaARK1) is a prototypic member of the GRK family that targets and phosphorylates agonist-occupied G protein-coupled receptors which causes functional uncoupling and a dampening of signaling, a process termed desensitization. There is a growing body of evidence demonstrating that the actions of GRKs in the heart are extremely important in modulating myocardial adrenergic signaling and hence cardiac function. Of particular interest to my laboratory, are the biochemical and physiological consequences of altering myocardial AR and GRK signaling. To address this, we are utilizing several in vitro and in vivo model systems. Of special interest, are the endogenous alterations that occur in myocardial GRKs and ARs during cardiovascular diseases such as heart failure (HF). We are also interested in the role of adrenergic signaling in the transplanted heart.
Since heart disease accounts for nearly 40% of all deaths annually in this country, it is of importance to learn more of the molecular pathology present in diseased myocardium. For example, in human HF regardless of the cause, a specific constellation of biochemical defects in cardiac tissue has been noted including loss of specific betaAR density and uncoupling of remaining receptors. Moreover, it has been shown that the levels of betaARK1 rise 3-5 fold in human HF, probably contributing to the dysfunction. Interestingly, it has recently been demonstrated that cardiovascular GRK levels (i.e. betaARK1) and activity were also increased in other disorders including myocardial hypertrophy and hypertension. Thus, a primary objective of my laboratory is to investigate the molecular mechanisms involved in myocardial GRK alterations and how these changes relate to adrenergic signaling. This research includes studying and exploring novel protein binding partners of GRKs in the myocardium using a variety of molecular biochemical assays including GRK-affinity columns.
Over the last several years, my laboratory has been investigating the consequences of altering myocardial ARs and GRKs in several in vivo model systems. Original studies were done in transgenic mice where AR and GRK-based transgenes were targeted specifically to the heart. These studies have been done in close collaboration with Dr. Robert Lefkowitz and Dr. Howard Rockman here at Duke University. We have found that altering AR signaling in the hearts of transgenic mice has profound effects on cardiovascular physiology including the findings that overexpression of beta2ARs or a peptide inhibitor of betaARK1 known as the betaARKct, significantly enhances in vivo cardiac contractility. We now have transgenic animals with myocardial-targeted overexpression of several ARs (alpha and beta) or GRKs and are studying the specific in vivo interactions of these molecules in the heart. Moreover, in collaboration with Drs. Lefkowitz and Rockman, we are generating a host of tissue and temporal specific conditional-knockout mice targeting the myocardial betaAR and GRK systems. Another area of transgenic animals being studied exclusively in my laboratory is altered betaAR and GRK activity specifically targeted to arterial smooth muscle in order to determine the role of AR signaling and desensitization in hypertension and other vascular diseases. These mice have recently been generated in our laboratory and are the target of current investigations.
Over the last year, we have been able to specifically study the role of betaAR desensitization and the role of betaARK1 in HF using genetically engineered mouse models of cardiomyopathy and HF. These studies have lead to the findings that inhibition of betaARK1 through myocardial-targeted betaARKct expression has rescued three separate genetic mouse models of cardiomyopathy, preventing the development of HF. The molecular study of these genetic models through DNA array technology is a logical step in the evolution of these studies and this is a new area of focus in the laboratory. Thus, we are analyzing the hearts of mice with altered betaAR and/or betaARK1 signaling by gene (DNA) chips to determine other genes that have been induced or silenced by our transgenes or gene knockouts. We are excited about using this technology in the laboratory taking advantage of our novel mouse models. Specifically, comparing differential gene expression in a failing mouse heart and comparing the genetic pattern in a heart that has been "rescued" by the betaARKct could lead to the elucidation of specific genes involved in the pathogenesis of HF. Importantly, this may also lead to novel therapeutic approaches to treating this disease as well as other cardiovascular disorders.
Our findings in mice that show that we can genetically enhance the functional contractility of the heart form the basis of another major focus of my research program which is the investigation of enhancing the in vivo function of the compromised heart via acute genetic manipulation using gene therapy. My laboratory heads the Cardiovascular Gene Therapy Program at Duke University Medical Center. The ability to manipulate betaAR density or receptor desensitization in the diseased heart is of great interest since it may provide unique inotropic support and improve existing therapeutic strategies. Gene transfer to the heart in vivo is a powerful approach to study the specific role of GRKs and adrenergic desensitization in both normal and diseased myocardium. Currently, we are employing various in vivo gene delivery techniques using adenoviruses, in order to effectively express specific betaAR and GRK-based transgenes which may produce alterations in myocardial signaling and global cardiac function. We also have established several surgical models of compromised heart function to use in these gene transfer studies including experimental HF in rabbits and pigs, and cardiac transplantation models in rats and rabbits. In addition to studying the feasibility of gene therapy approaches to HF, the Cardiovascular Gene Therapy Program here at Duke has developed a molecular gene therapy strategy to prevention of pathological vascular smooth muscle intimal hyperplasia such as coronary artery restenosis after angioplasty. Our first clinical trial employing adenoviral-mediated gene therapy for restenosis is tentatively planned for the end of 2002.

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