Reversible lysine fatty acylation of an anchoring protein mediates adipocyte adrenergic signaling.

N-myristoylation on glycine is an irreversible modification that has long been recognized to govern protein localization and function. In contrast, the biological roles of lysine myristoylation remain ill-defined. We demonstrate that the cytoplasmic scaffolding protein, gravin-α/A kinase-anchoring protein 12, is myristoylated on two lysine residues embedded in its carboxyl-terminal protein kinase A (PKA) binding domain. Histone deacetylase 11 (HDAC11) docks to an adjacent region of gravin-α and demyristoylates these sites. In brown and white adipocytes, lysine myristoylation of gravin-α is required for signaling via ß2- and ß3-adrenergic receptors (ß-ARs), which are G protein-coupled receptors (GPCRs). Lysine myristoylation of gravin-α drives ß-ARs to lipid raft membrane microdomains, which results in PKA activation and downstream signaling that culminates in protective thermogenic gene expression. These findings define reversible lysine myristoylation as a mechanism for controlling GPCR signaling and highlight the potential of inhibiting HDAC11 to manipulate adipocyte phenotypes for therapeutic purposes.

Beyond the genome: challenges and potential for epigenetics-driven therapeutic approaches in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease of the cardiopulmonary system caused by the narrowing of the pulmonary arteries, leading to increased vascular resistance and pressure. This leads to right ventricle remodeling, dysfunction, and eventually, death. While conventional therapies have largely focused on targeting vasodilation, other pathological features of PAH including aberrant inflammation, mitochondrial dynamics, cell proliferation, and migration have not been well explored. Thus, despite some recent improvements in PAH treatment, the life expectancy and quality of life for patients with PAH remains poor. Showing many similarities to cancers, PAH is characterized by increased pulmonary arterial smooth muscle cell proliferation, decreased apoptotic signaling pathways, and changes in metabolism. The recent successes of therapies targeting epigenetic modifiers for the treatment of cancer has prompted epigenetic research in PAH, revealing many new potential therapeutic targets. In this minireview we discuss the emergence of epigenetic dysregulation in PAH and highlight epigenetic-targeting compounds that may be effective for the treatment of PAH.

Cross Section of Legislative Approaches to Reducing Indoor Dampness and Mold.

Exposure to indoor dampness and mold is associated with numerous adverse respiratory conditions, including asthma. While no quantitative health-based threshold currently exists for mold, the conditions that support excessive dampness and mold are known and preventable; experts agree that controlling these conditions could lead to substantial savings in health care costs and improvement in public health. This article reviews a sample of state and local policies to limit potentially harmful exposures. Adoption of laws to strengthen building codes, specify dampness and mold in habitability laws, regulate mold contractors, and other legislative approaches are discussed, as are key factors supporting successful implementation. Communicating these lessons learned could accelerate the process for other jurisdictions considering similar approaches. Information about effectiveness of legislation as prevention is lacking; thus, evaluation could yield important information to inform the development of model state or local laws that significantly address mold as a public health concern.

Interplay between the E2F pathway and ß-adrenergic signaling in the pathological hypertrophic response of myocardium.

The E2F/Pocket protein (Rb) pathway regulates cell growth, differentiation, and death by modulating gene expression. We previously examined this pathway in the myocardium via manipulation of the unique E2F repressor, E2F6, which is believed to repress gene activity independently of Rb. Mice with targeted expression of E2F6 in postnatal myocardium developed dilated cardiomyopathy (DCM) without hypertrophic growth. We assessed the mechanisms of the apparent failure of compensatory hypertrophic growth as well as their response to the ß-adrenergic agonist isoproterenol. As early as 2 weeks, E2F6 transgenic (Tg) mice present with dilated thinner left ventricles and significantly reduced ejection fraction and fractional shortening which persists at 6 weeks of age, but with no apparent increase in left ventricle weight: body weight (LVW:BW). E2F6-Tg mice treated with isoproterenol (6.1 mg/kg/day) show double the increase in LVW:BW than their Wt counterparts (32% vs 16%, p-value: 0.007). Western blot analysis revealed the activation of the adrenergic pathway in Tg heart tissue under basal conditions with ~2-fold increase in the level of ß2-adrenergic receptors (p-value: 8.9E-05), protein kinase A catalytic subunit (PKA-C) (p-value: 0.0176), activated c-Src tyrosine-protein kinase (p-value: 0.0002), extracellular receptor kinase 2 (ERK2) (p-value: 0.0005), and induction of the anti-apoptotic protein Bcl2 (p-value 0. 0.00001). In contrast, a ~60% decrease in the cardiac growth regulator: AKT1 (p-value 0.0001) and a ~four fold increase in cyclic AMP dependent phosphodiesterase 4D (PDE4D), the negative regulator of PKA activity, were evident in the myocardium of E2F6-Tg mice. The expression of E2F3 was down-regulated by E2F6, but was restored by isoproterenol. Further, Rb expression was down-regulated in Tg mice in response to isoproterenol implying a net activation of the E2F pathway. Thus the unique regulation of E2F activity by E2F6 renders the myocardium hypersensitive to adrenergic stimulus resulting in robust hypertrophic growth. These data reveal a novel interplay between the E2F pathway, ß2-adrenergic/PKA/PDE4D, and ERK/c-Src axis in fine tuning the pathological hypertrophic growth response. E2F6 deregulates E2F3 such that pro-hypertrophic growth and survival are enhanced via ß2-adrenergic signaling however this response is outweighed by the induction of anti-hypertrophic signals so that left ventricle dilation proceeds without any increase in muscle mass.

The E2F6 repressor activates gene expression in myocardium resulting in dilated cardiomyopathy.

The E2F/Rb pathway regulates cardiac growth and development and holds great potential as a therapeutic target. The E2F6 repressor is a unique E2F member that acts independently of pocket proteins. Forced expression of E2F6 in mouse myocardium induced heart failure and mortality, with severity of symptoms correlating to E2F6 levels. Echocardiography demonstrated a 37% increase (P<0.05) in left ventricular end-diastolic diameter and reduced ejection fraction (<40%, P<0.05) in young transgenic (Tg) mice. Microarray and qPCR analysis revealed a paradoxical increase in E2F-responsive genes, which regulate the cell cycle, without changes in cardiomyocyte cell number or size in Tg mice. Young adult Tg mice displayed a 75% (P<0.01) decrease in gap junction protein connexin-43, resulting in abnormal electrocardiogram including a 24% (P<0.05) increase in PR interval. Further, mir-206, which targets connexin-43, was up-regulated 10-fold (P<0.05) in Tg myocardium. The mitogen-activated protein kinase pathway, which regulates the levels of miR-206 and connexin-43, was activated in Tg hearts. Thus, deregulated E2F6 levels evoked abnormal gene expression at transcriptional and post-transcriptional levels, leading to cardiac remodeling and dilated cardiomyopathy. The data highlight an unprecedented role for the strict regulation of the E2F pathway in normal postnatal cardiac function.

HDAC11 inhibition triggers bimodal thermogenic pathways to circumvent adipocyte catecholamine resistance.

Stimulation of adipocyte ß-adrenergic receptors (ß-ARs) induces expression of uncoupling protein 1 (UCP1), promoting nonshivering thermogenesis. Association of ß-ARs with a lysine-myristoylated form of A kinase-anchoring protein 12 (AKAP12, also known as gravin-α) is required for downstream signaling that culminates in UCP1 induction. Conversely, demyristoylation of gravin-α by histone deacetylase 11 (HDAC11) suppresses this pathway. Whether inhibition of HDAC11 in adipocytes is sufficient to drive UCP1 expression independently of ß-ARs is not known. Here, we demonstrate that adipocyte-specific deletion of HDAC11 in mice leads to robust induction of UCP1 in adipose tissue (AT), resulting in increased body temperature. These effects are mimicked by treating mice in vivo or human AT ex vivo with an HDAC11-selective inhibitor, FT895. FT895 triggers biphasic, gravin-α myristoylation-dependent induction of UCP1 protein expression, with a noncanonical acute response that is posttranscriptional and independent of protein kinase A (PKA), and a delayed response requiring PKA activity and new Ucp1 mRNA synthesis. Remarkably, HDAC11 inhibition promotes UCP1 expression even in models of adipocyte catecholamine resistance where ß-AR signaling is blocked. These findings define cell-autonomous, multimodal roles for HDAC11 as a suppressor of thermogenesis, and highlight the potential of inhibiting HDAC11 to therapeutically alter AT phenotype independently of ß-AR stimulation.

HDAC11 inhibition triggers bimodal thermogenic pathways to circumvent adipocyte catecholamine resistance.

Stimulation of adipocyte ß-adrenergic receptors (ß-ARs) induces expression of uncoupling protein 1 (UCP1), promoting non-shivering thermogenesis. Association of ß-ARs with a lysine myristoylated form of A-kinase anchoring protein 12 (AKAP12)/gravin-α is required for downstream signaling that culminates in UCP1 induction. Conversely, demyristoylation of gravin-α by histone deacetylase 11 (HDAC11) suppresses this pathway. Whether inhibition of HDAC11 in adipocytes is sufficient to drive UCP1 expression independently of ß-ARs is not known. Here, we demonstrate that adipocyte-specific deletion of HDAC11 in mice leads to robust induction of UCP1 in adipose tissue (AT), resulting in increased body temperature. These effects are mimicked by treating mice in vivo or human AT ex vivo with an HDAC11-selective inhibitor, FT895. FT895 triggers biphasic, gravin-α myristoylation-dependent induction of UCP1 protein expression, with a non-canonical acute response that is post-transcriptional and independent of protein kinase A (PKA), and a delayed response requiring PKA activity and new Ucp1 mRNA synthesis. Remarkably, HDAC11 inhibition promotes UCP1 expression even in models of adipocyte catecholamine resistance where ß-AR signaling is blocked. These findings define cell autonomous, multi-modal roles for HDAC11 as a suppressor of thermogenesis, and highlight the potential of inhibiting HDAC11 to therapeutically alter AT phenotype independently of ß-AR stimulation.

HDAC6 modulates myofibril stiffness and diastolic function of the heart.

Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.

Application of microRNA Database Mining in Biomarker Discovery and Identification of Therapeutic Targets for Complex Disease.

Over the past two decades, it has become increasingly evident that microRNAs (miRNA) play a major role in human diseases such as cancer and cardiovascular diseases. Moreover, their easy detection in circulation has made them a tantalizing target for biomarkers of disease. This surge in interest has led to the accumulation of a vast amount of miRNA expression data, prediction tools, and repositories. We used the Human microRNA Disease Database (HMDD) to discover miRNAs which shared expression patterns in the related diseases of ischemia/reperfusion injury, coronary artery disease, stroke, and obesity as a model to identify miRNA candidates for biomarker and/or therapeutic intervention in complex human diseases. Our analysis identified a single miRNA, hsa-miR-21, which was casually linked to all four pathologies, and numerous others which have been detected in the circulation in more than one of the diseases. Target analysis revealed that hsa-miR-21 can regulate a number of genes related to inflammation and cell growth/death which are major underlying mechanisms of these related diseases. Our study demonstrates a model for researchers to use HMDD in combination with gene analysis tools to identify miRNAs which could serve as biomarkers and/or therapeutic targets of complex human diseases.

The Brain-Heart Axis: Alzheimer's, Diabetes, and Hypertension.

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder affecting millions worldwide. Currently, there are only four approved treatments for AD, which improve symptoms modestly. AD is believed to be caused by the formation of intercellular plaques and intracellular tangles in the brain, but thus far all new drugs which target these pathologies have failed clinical trials. New research highlights the link between AD and Type II Diabetes (T2D), and some believe that AD is actually a brain specific form of it termed Type III Diabetes (T3D). Drugs which are currently approved for the treatment of T2D, such as metformin, have shown promising results in improving cognitive function and even preventing the development of AD in diabetic patients. Recent studies shed light on the relationship between the brain and cardiovascular system in which the brain and heart communicate with one another via the vasculature to regulate fluid and nutrient homeostasis. This line of research reveals how the brain-heart axis regulates hypertension and diabetes, both of which can impact cognitive function. In this review we survey past and ongoing research and clinical trials for AD, and argue that AD is a complex and systemic disorder which requires comprehensive approaches beyond the brain for effective prevention and/or treatment.

SLMAP3 isoform modulates cardiac gene expression and function.

The sarcolemmal membrane associated proteins (SLMAPs) belong to the super family of tail anchored membrane proteins which serve diverse roles in biology including cell growth, protein trafficking and ion channel regulation. Mutations in human SLMAP have been linked to Brugada syndrome with putative deficits in trafficking of the sodium channel (Nav1.5) to the cell membrane resulting in aberrant electrical activity and heart function. Three main SLMAP isoforms (SLMAP1 (35 kDa), SLMAP2 (45 kDa), and SLMAP3 (91 kDa)) are expressed in myocardium but their precise role remains to be defined. Here we generated transgenic (Tg) mice with cardiac-specific expression of the SLMAP3 isoform during postnatal development which present with a significant decrease (20%) in fractional shortening and (11%) in cardiac output at 5 weeks of age. There was a lack of any notable cardiac remodeling (hypertrophy, fibrosis or fetal gene activation) in Tg hearts but the electrocardiogram indicated a significant increase (14%) in the PR interval and a decrease (43%) in the R amplitude. Western blot analysis indicated a selective and significant decrease (55%) in protein levels of Nav1.5 while 45% drop in its transcript levels were detectable by qRT-PCR. Significant decreases in the protein and transcript levels of the calcium transport system of the sarcoplasmic reticulum (SERCA2a/PLN) were also evident in Tg hearts. These data reveal a novel role for SLMAP3 in the selective regulation of important ion transport proteins at the level of gene expression and suggest that it may be a unique target in cardiovascular function and disease.

E2F6 protein levels modulate drug induced apoptosis in cardiomyocytes.

The E2F/Rb pathway regulates cell growth, differentiation, and death. In particular, E2F1 promotes apoptosis in all cells including those of the heart. E2F6, which represses E2F activity, was found to induce dilated cardiomyopathy in the absence of apoptosis in murine post-natal heart. Here we evaluate the anti-apoptotic potential of E2F6 in neonatal cardiomyocytes (NCM) from E2F6-Tg hearts which showed significantly less caspase-3 cleavage, a lower Bax/Bcl2 ratio, and improved cell viability in response to CoCl2 exposure. This correlated with a decrease in the pro-apoptotic E2F3 protein levels. In contrast, no difference in apoptotic markers or cell viability was observed in response to Doxorubicin (Dox) treatment between Wt and Tg-NCM. Dox caused a rapid and dramatic loss of the E2F6 protein in Tg-NCM within 6h and was undetectable after 12h. The level of e2f6 transcript was unchanged in Wt NCM, but was dramatically decreased in Tg cells in response to both Dox and CoCl2. This was related to an impact of the drugs on the α-myosin heavy chain promoter used to drive the E2F6 transgene. By comparison in HeLa, Dox induced apoptosis through upregulation of endogenous E2F1 involving post-transcriptional mechanisms, while E2F6 was down regulated with induction of the Checkpoint kinase-1 and proteasome degradation. These data imply that E2F6 serves to modulate E2F activity and protect cells including cardiomyocytes from apoptosis and improve survival. Strategies to modulate E2F6 levels may be therapeutically useful to mitigate cell death associated disorders.

E2F6 Impairs Glycolysis and Activates BDH1 Expression Prior to Dilated Cardiomyopathy.

RATIONALE: The E2F pathway plays a critical role in cardiac growth and development, yet its role in cardiac metabolism remains to be defined. Metabolic changes play important roles in human heart failure and studies imply the ketogenic enzyme ß-hydroxybutyrate dehydrogenase I (BDH1) is a potential biomarker. OBJECTIVE: To define the role of the E2F pathway in cardiac metabolism and dilated cardiomyopathy (DCM) with a focus on BDH1. METHODS AND RESULTS: We previously developed transgenic (Tg) mice expressing the transcriptional repressor, E2F6, to interfere with the E2F/Rb pathway in post-natal myocardium. These Tg mice present with an E2F6 dose dependent DCM and deregulated connexin-43 (CX-43) levels in myocardium. Using the Seahorse platform, a 22% decrease in glycolysis was noted in neonatal cardiomyocytes isolated from E2F6-Tg hearts. This was associated with a 39% reduction in the glucose transporter GLUT4 and 50% less activation of the regulator of glucose metabolism AKT2. The specific reduction of cyclin B1 (70%) in Tg myocardium implicates its importance in supporting glycolysis in the postnatal heart. No changes in cyclin D expression (known to regulate mitochondrial activity) were noted and lipid metabolism remained unchanged in neonatal cardiomyocytes from Tg hearts. However, E2F6 induced a 40-fold increase of the Bdh1 transcript and 890% increase in its protein levels in hearts from Tg pups implying a potential impact on ketolysis. By contrast, BDH1 expression is not activated until adulthood in normal myocardium. Neonatal cardiomyocytes from Wt hearts incubated with the ketone ß-hydroxybutyrate (ß-OHB) showed a 100% increase in CX-43 protein levels, implying a role for ketone signaling in gap junction biology. Neonatal cardiomyocyte cultures from Tg hearts exhibited enhanced levels of BDH1 and CX-43 and were not responsive to ß-OHB. CONCLUSIONS: The data reveal a novel role for the E2F pathway in regulating glycolysis in the developing myocardium through a mechanism involving cyclin B1. We reveal BDH1 expression as an early biomarker of heart failure and its potential impact, through ketone signaling, on CX-43 levels in E2F6-induced DCM.