Kyoung-Han Kim

Obesity-associated variants within FTO form long-range functional connections with IRX3

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.

FTO Obesity Variant Circuitry and Adipocyte Browning in Humans

BACKGROUND Genomewide association studies can be used to identify disease-relevant genomic regions, but interpretation of the data is challenging. The FTO region harbors the strongest genetic association with obesity, yet the mechanistic basis of this association remains elusive. METHODS We examined epigenomic data, allelic activity, motif conservation, regulator expression, and gene coexpression patterns, with the aim of dissecting the regulatory circuitry and mechanistic basis of the association between the FTO region and obesity. We validated our predictions with the use of directed perturbations in samples from patients and from mice and with endogenous CRISPR-Cas9 genome editing in samples from patients. RESULTS Our data indicate that the FTO allele associated with obesity represses mitochondrial thermogenesis in adipocyte precursor cells in a tissue-autonomous manner. The rs1421085 T-to-C single-nucleotide variant disrupts a conserved motif for the ARID5B repressor, which leads to derepression of a potent preadipocyte enhancer and a doubling of IRX3 and IRX5 expression during early adipocyte differentiation. This results in a cell-autonomous developmental shift from energy-dissipating beige (brite) adipocytes to energy-storing white adipocytes, with a reduction in mitochondrial thermogenesis by a factor of 5, as well as an increase in lipid storage. Inhibition of Irx3 in adipose tissue in mice reduced body weight and increased energy dissipation without a change in physical activity or appetite. Knockdown of IRX3 or IRX5 in primary adipocytes from participants with the risk allele restored thermogenesis, increasing it by a factor of 7, and overexpression of these genes had the opposite effect in adipocytes from nonrisk-allele carriers. Repair of the ARID5B motif by CRISPR-Cas9 editing of rs1421085 in primary adipocytes from a patient with the risk allele restored IRX3 and IRX5 repression, activated browning expression programs, and restored thermogenesis, increasing it by a factor of 7. CONCLUSIONS Our results point to a pathway for adipocyte thermogenesis regulation involving ARID5B, rs1421085, IRX3, and IRX5, which, when manipulated, had pronounced pro-obesity and anti-obesity effects. (Funded by the German Research Center for Environmental Health and others.).

The Homeodomain Transcription Factor Irx5 Establishes the Mouse Cardiac Ventricular Repolarization Gradient

Rhythmic cardiac contractions depend on the organized propagation of depolarizing and repolarizing wavefronts. Repolarization is spatially heterogeneous and depends largely on gradients of potassium currents. Gradient disruption in heart disease may underlie susceptibility to fatal arrhythmias, but it is not known how this gradient is established. We show that, in mice lacking the homeodomain transcription factor Irx5, the cardiac repolarization gradient is abolished due to increased Kv4.2 potassium-channel expression in endocardial myocardium, resulting in a selective increase of the major cardiac repolarization current, I(to,f), and increased susceptibility to arrhythmias. Myocardial Irx5 is expressed in a gradient opposite that of Kv4.2, and Irx5 represses Kv4.2 expression by recruiting mBop, a cardiac transcriptional repressor. Thus, an Irx5 repressor gradient negatively regulates potassium-channel-gene expression in the heart, forming an inverse I(to,f) gradient that ensures coordinated cardiac repolarization while also preventing arrhythmias.

Glucagon-Like Peptide (GLP)-1(9-36)Amide-Mediated Cytoprotection Is Blocked by Exendin(9-39) Yet Does Not Require the Known GLP-1 Receptor

The widely expressed dipeptidyl peptidase-4 enzyme rapidly cleaves the gut hormone glucagon-like peptide-1 [GLP-1(7-36)amide] at the N terminus to generate GLP-1(9-36)amide. Both intact GLP-1(7-36)amide and GLP-1(9-36)amide exert cardioprotective actions in rodent hearts; however, the mechanisms underlying the actions of GLP-1(9-36)amide remain poorly understood. We used mass spectrometry of coronary effluents to demonstrate that isolated mouse hearts rapidly convert infused GLP-1(7-36)amide to GLP-1(9-36)amide. After ischemia-reperfusion (I/R) injury of isolated mouse hearts, administration of GLP-1(9-36)amide or exendin-4 improved functional recovery and reduced infarct size. The direct actions of these peptides were studied in cultured neonatal mouse cardiomyocytes. Both GLP-1(9-36)amide and exendin-4 increased levels of cAMP and phosphorylation of ERK1/2 and the phosphoinositide 3-kinase target protein kinase B/Akt. In I/R injury models in vitro, both peptides improved mouse cardiomyocyte viability and reduced lactate dehydrogenase release and caspase-3 activation. These effects were attenuated by inhibitors of ERK1/2 and phosphoinositide 3-kinase. Unexpectedly, the cardioprotective actions of GLP-1(9-36)amide were blocked by exendin(9-39) yet preserved in Glp1r(-/-) cardiomyocytes. Furthermore, GLP-1(9-36)amide, but not exendin-4, improved the survival of human aortic endothelial cells undergoing I/R injury, actions sensitive to the nitric oxide synthase inhibitor, N(G)-nitro-l-arginine methyl ester (L-NAME). In summary, our findings demonstrate separate actions for GLP-1(9-36)amide vs. the GLP-1R agonist exendin-4 and reveal the existence of a GLP-1(9-36)amide-responsive, exendin(9-39)-sensitive, cardioprotective signaling pathway distinct from that associated with the classical GLP-1 receptor.

MEK-ERK pathway modulation ameliorates disease phenotypes in a mouse model of Noonan syndrome associated with the Raf1(L613V) mutation.

Hypertrophic cardiomyopathy (HCM) is a leading cause of sudden death in children and young adults. Abnormalities in several signaling pathways are implicated in the pathogenesis of HCM, but the role of the RAS-RAF-MEK-ERK MAPK pathway has been controversial. Noonan syndrome (NS) is one of several autosomal-dominant conditions known as RASopathies, which are caused by mutations in different components of this pathway. Germline mutations in RAF1 (which encodes the serine-threonine kinase RAF1) account for approximately 3%-5% of cases of NS. Unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with HCM. To explore the pathogenesis of such mutations, we generated knockin mice expressing the NS-associated Raf1(L613V) mutation. Like NS patients, mice heterozygous for this mutation (referred to herein as L613V/+ mice) had short stature, craniofacial dysmorphia, and hematologic abnormalities. Valvuloseptal development was normal, but L613V/+ mice exhibited eccentric cardiac hypertrophy and aberrant cardiac fetal gene expression, and decompensated following pressure overload. Agonist-evoked MEK-ERK activation was enhanced in multiple cell types, and postnatal MEK inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice. These data show that different NS genes have intrinsically distinct pathological effects, demonstrate that enhanced MEK-ERK activity is critical for causing HCM and other RAF1-mutant NS phenotypes, and suggest a mutation-specific approach to the treatment of RASopathies.

Erythropoietin Protects against Doxorubicin-Induced Cardiomyopathy via a Phosphatidylinositol 3-Kinase-Dependent Pathway

Doxorubicin (DOX) is an effective antineoplastic agent whose use has been limited by its cardiotoxic side effects. Recent studies have established that erythropoietin (EPO), a cytokine essential for red blood cell production, protects against ischemic injury in the heart and other organs. The purpose of this study was to assess whether EPO protects the heart against cardiotoxicity induced by DOX. We found that DOX-induced apoptosis and impaired heart function in mice were largely prevented by EPO administration. To investigate the mechanism of protection by EPO, cultured neonatal mouse ventricular myocytes were treated with EPO at therapeutic levels (i.e., 1 U/ml), before application of DOX (0.1–1.0 μM). EPO protected against DOX-induced cardiomyocyte death (by ≈50%) and apoptosis assessed by annexin-V labeling, DNA fragmentation, and caspase-3 activity. DOX-mediated increases in reactive oxygen species, which trigger cardiotoxicity, were also reversed by preconditioning with EPO. These functional effects of EPO correlated with increased Akt/protein kinase B (∼2-fold) and glycogen synthase kinase 3 (GSK-3; ∼1.3-fold) phosphorylations, suggesting protection by EPO was mediated by phosphatidylinositol 3-kinase activation. Indeed, preventing Akt and GSK-3β phosphorylations by phosphatidylinositol 3-kinase (PI3K) inhibition abolished protection by EPO against cardiomyocyte loss, apoptosis, and oxidative stress. Thus, pretreatment with therapeutic levels of EPO can protect the myocardium against DOX-induced impaired heart function and cardiomyocyte apoptosis by activating PI3K-Akt cell survival pathways.

Iroquois homeobox gene 3 establishes fast conduction in the cardiac His–Purkinje network

Rapid electrical conduction in the His–Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His–Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction–forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for Irx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart.

Constitutively active calcineurin induces cardiac endoplasmic reticulum stress and protects against apoptosis that is mediated by α-crystallin-B

Cardiac-specific overexpression of a constitutively active form of calcineurin A (CNA) leads directly to cardiac hypertrophy in the CNA mouse model. Because cardiac hypertrophy is a prominent characteristic of many cardiomyopathies, we deduced that delineating the proteomic profile of ventricular tissue from this model might identify novel, widely applicable therapeutic targets. Proteomic analysis was carried out by subjecting fractionated cardiac samples from CNA mice and their WT littermates to gel-free liquid chromatography linked to shotgun tandem mass spectrometry. We identified 1,918 proteins with high confidence, of which 290 were differentially expressed. Microarray analysis of the same tissue provided us with alterations in the ventricular transcriptome. Because bioinformatic analyses of both the proteome and transcriptome demonstrated the up-regulation of endoplasmic reticulum stress, we validated its occurrence in adult CNA hearts through a series of immunoblots and RT-PCR analyses. Endoplasmic reticulum stress often leads to increased apoptosis, but apoptosis was minimal in CNA hearts, suggesting that activated calcineurin might protect against apoptosis. Indeed, the viability of cultured neonatal mouse cardiomyocytes (NCMs) from CNA mice was higher than WT after serum starvation, an apoptotic trigger. Proteomic data identified α-crystallin B (Cryab) as a potential mediator of this protective effect and we showed that silencing of Cryab via lentivector-mediated transduction of shRNAs in NCMs led to a significant reduction in NCM viability and loss of protection against apoptosis. The identification of Cryab as a downstream effector of calcineurin-induced protection against apoptosis will permit elucidation of its role in cardiac apoptosis and its potential as a therapeutic target.

Cooperative and antagonistic roles for Irx3 and Irx5 in cardiac morphogenesis and postnatal physiology

The Iroquois homeobox (Irx) homeodomain transcription factors are important for several aspects of embryonic development. In the developing heart, individual Irx genes are important for certain postnatal cardiac functions, including cardiac repolarization (Irx5) and rapid ventricular conduction (Irx3). Irx genes are expressed in dynamic and partially overlapping patterns in the developing heart. Here we show in mice that Irx3 and Irx5 have redundant function in the endocardium to regulate atrioventricular canal morphogenesis and outflow tract formation. Our data suggest that direct transcriptional repression of Bmp10 by Irx3 and Irx5 in the endocardium is required for ventricular septation. A postnatal deletion of Irx3 and Irx5 in the myocardium leads to prolongation of atrioventricular conduction, due in part to activation of expression of the Na+ channel protein Nav1.5. Surprisingly, combined postnatal loss of Irx3 and Irx5 results in a restoration of the repolarization gradient that is altered in Irx5 mutant hearts, suggesting that postnatal Irx3 activity can be repressed by Irx5. Our results have uncovered complex genetic interactions between Irx3 and Irx5 in embryonic cardiac development and postnatal physiology.

Iroquois Homeodomain Transcription Factors in Heart Development and Function

Numerous cardiac transcription factors play overlapping roles in both the specification and proliferation of the cardiac tissues and chambers during heart development. It has become increasingly apparent that cardiac transcription factors also play critical roles in the regulation of expression of many functional genes in the prenatal and postnatal hearts. Accordingly, mutations of cardiac transcription factors cannot only result in congenital heart defects but also alter heart function thereby predisposing to heart disease and cardiac arrhythmias. In this review, we summarize the roles of Iroquois homeobox (Irx) family of transcription factors in heart development and function. In all, 6 Irx genes are expressed with distinct and overlapping patterns in the mammalian heart. Studies in several animal models demonstrate that Irx genes are important for the establishment of ventricular chamber properties, the ventricular conduction system, as well as heterogeneity of the ventricular repolarization. The molecular mechanisms by which Irx proteins regulate gene expression and the clinical relevance of Irx functions in the heart are discussed.