Manveen K. Gupta

Unique microRNA profile in end-stage heart failure indicates alterations in specific cardiovascular signaling networks.

It is well established that gene expression patterns are substantially altered in cardiac hypertrophy and heart failure, but the reasons for such differences are not clear. MicroRNAs (miRNAs) are short noncoding RNAs that provide a novel mechanism for gene regulation. The goal of this study was to comprehensively test for alterations in miRNA expression using human heart failure samples with an aim to build signaling pathway networks using predicted targets for the miRNAs and to identify nodal molecules that control these networks. Genome-wide profiling of miRNAs was performed using custom-designed miRNA microarray followed by validation on an independent set of samples. Eight miRNAs are significantly altered in heart failure of which we have identified two novel miRNAs that are yet to be implicated in cardiac pathophysiology. To gain an unbiased global perspective on regulation by altered miRNAs, predicted targets of eight miRNAs were analyzed using the Ingenuity Pathways Analysis network algorithm to build signaling networks and identify nodal molecules. The majority of nodal molecules identified in our analysis are targets of altered miRNAs and are known regulators of cardiovascular signaling. A heart failure gene expression data base was used to analyze changes in expression patterns for these target nodal molecules. Indeed, expression of nodal molecules was altered in heart failure and inversely correlated to miRNA changes validating our analysis. Importantly, using network analysis we have identified a limited number of key functional targets that may regulate expression of the myriad proteins in heart failure and could be potential therapeutic targets.

Long-term α1A-adrenergic receptor stimulation improves synaptic plasticity, cognitive function, mood, and longevity.

The role of α1-adrenergic receptors (α1ARs) in cognition and mood is controversial, probably as a result of past use of nonselective agents. α1AAR activation was recently shown to increase neurogenesis, which is linked to cognition and mood. We studied the effects of long-term α1AAR stimulation using transgenic mice engineered to express a constitutively active mutant (CAM) form of the α1AAR. CAM-α1AAR mice showed enhancements in several behavioral models of learning and memory. In contrast, mice that have the α1AAR gene knocked out displayed poor cognitive function. Hippocampal brain slices from CAM-α1AAR mice demonstrated increased basal synaptic transmission, paired-pulse facilitation, and long-term potentiation compared with wild-type (WT) mice. WT mice treated with the α1AAR-selective agonist cirazoline also showed enhanced cognitive functions. In addition, CAM-α1AAR mice exhibited antidepressant and less anxious phenotypes in several behavioral tests compared with WT mice. Furthermore, the lifespan of CAM-α1AAR mice was 10% longer than that of WT mice. Our results suggest that long-term α1AAR stimulation improves synaptic plasticity, cognitive function, mood, and longevity. This may afford a potential therapeutic target for counteracting the decline in cognitive function and mood associated with aging and neurological disorders.

α1-Adrenergic Receptors Regulate Neurogenesis and Gliogenesis

The understanding of the function of α1-adrenergic receptors in the brain has been limited due to a lack of specific ligands and antibodies. We circumvented this problem by using transgenic mice engineered to overexpress either wild-type receptor tagged with enhanced green fluorescent protein or constitutively active mutant α1-adrenergic receptor subtypes in tissues in which they are normally expressed. We identified intriguing α1A-adrenergic receptor subtype-expressing cells with a migratory morphology in the adult subventricular zone that coexpressed markers of neural stem cell and/or progenitors. Incorporation of 5-bromo-2-deoxyuridine in vivo increased in neurogenic areas in adult α1A-adrenergic receptor transgenic mice or normal mice given the α1A-adrenergic receptor-selective agonist, cirazoline. Neonatal neurospheres isolated from normal mice expressed a mixture of α1-adrenergic receptor subtypes, and stimulation of these receptors resulted in increased expression of the α1B-adrenergic receptor subtype, proneural basic helix-loop-helix transcription factors, and the differentiation and migration of neuronal progenitors for catecholaminergic neurons and interneurons. α1-Adrenergic receptor stimulation increased the apoptosis of astrocytes and regulated survival of neonatal neurons through phosphatidylinositol 3-kinase signaling. However, in adult normal neurospheres, α1-adrenergic receptor stimulation increased the expression of glial markers at the expense of neuronal differentiation. In vivo, S100-positive glial and βIII tubulin neuronal progenitors colocalized with either α1-adrenergic receptor subtype in the olfactory bulb. Our results indicate that α1-adrenergic receptors can regulate both neurogenesis and gliogenesis that may be developmentally dependent. Our findings may lead to new therapies to treat neurodegenerative diseases.

miRNA-548c: A specific signature in circulating PBMCs from dilated cardiomyopathy patients

High fidelity genome-wide expression analysis has strengthened the idea that microRNA (miRNA) signatures in peripheral blood mononuclear cells (PBMCs) can be potentially used to predict the pathology when anatomical samples are inaccessible like the heart. PBMCs from 48 non-failing controls and 44 patients with relatively stable chronic heart failure (ejection fraction of ≤ 40%) associated with dilated cardiomyopathy (DCM) were used for miRNA analysis. Genome-wide miRNA-microarray on PBMCs from chronic heart failure patients identified miRNA signature uniquely characterized by the downregulation of miRNA-548 family members. We have also independently validated downregulation of miRNA-548 family members (miRNA-548c & 548i) using real time-PCR in a large cohort of independent patient samples. Independent in silico Ingenuity Pathway Analysis (IPA) of miRNA-548 targets shows unique enrichment of signaling molecules and pathways associated with cardiovascular disease and hypertrophy. Consistent with specificity of miRNA changes with pathology, PBMCs from breast cancer patients showed no alterations in miRNA-548c expression compared to healthy controls. These studies suggest that miRNA-548 family signature in PBMCs can therefore be used to detect early heart failure. Our studies show that cognate networking of predicted miRNA-548 targets in heart failure can be used as a powerful ancillary tool to predict the ongoing pathology.

Gβγ-Independent Recruitment of G-Protein Coupled Receptor Kinase 2 Drives Tumor Necrosis Factor α–Induced Cardiac β-Adrenergic Receptor Dysfunction

Background— Proinflammatory cytokine tumor necrosis factor-&agr; (TNF&agr;) induces &bgr;-adrenergic receptor (&bgr;AR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. Methods and Results— Two different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNF&agr; showed that TNF&agr; alone is sufficient to mediate &bgr;AR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling &bgr;AR desensitization independent of sympathetic overdrive. TNF&agr;-mediated &bgr;AR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in &bgr;2AR-overexpressing human embryonic kidney 293 cells showed significant &bgr;AR desensitization, GRK2 upregulation, and recruitment to the &bgr;AR complex following TNF&agr;. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated &bgr;AR phosphorylation and GRK2 recruitment on TNF&agr;. Furthermore, TNF&agr;-mediated &bgr;AR phosphorylation was not blocked with &bgr;AR antagonist propranolol. Additionally, TNF&agr; administration in transgenic mice with cardiac overexpression of G&bgr;&ggr;-sequestering peptide &bgr;ARK-ct could not prevent &bgr;AR desensitization or cardiac dysfunction showing that GRK2 recruitment to the &bgr;AR is G&bgr;&ggr; independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNF&agr;-mediated &bgr;AR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNF&agr;-mediated loss in contractility, showing that TNF&agr;-induced &bgr;AR desensitization is GRK2 dependent. Conclusions— TNF&agr;-induced &bgr;AR desensitization is mediated by GRK2 and is independent of G&bgr;&ggr;, uncovering a hitherto unknown cross-talk between TNF&agr; and &bgr;AR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.

Gβγ Independent Recruitment of G-Protein Coupled Receptor Kinase 2 Drives TNFα-Induced Cardiac Beta-Adrenergic Receptor Dysfunction

Background— Proinflammatory cytokine tumor necrosis factor-&agr; (TNF&agr;) induces &bgr;-adrenergic receptor (&bgr;AR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. Methods and Results— Two different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNF&agr; showed that TNF&agr; alone is sufficient to mediate &bgr;AR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling &bgr;AR desensitization independent of sympathetic overdrive. TNF&agr;-mediated &bgr;AR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in &bgr;2AR-overexpressing human embryonic kidney 293 cells showed significant &bgr;AR desensitization, GRK2 upregulation, and recruitment to the &bgr;AR complex following TNF&agr;. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated &bgr;AR phosphorylation and GRK2 recruitment on TNF&agr;. Furthermore, TNF&agr;-mediated &bgr;AR phosphorylation was not blocked with &bgr;AR antagonist propranolol. Additionally, TNF&agr; administration in transgenic mice with cardiac overexpression of G&bgr;&ggr;-sequestering peptide &bgr;ARK-ct could not prevent &bgr;AR desensitization or cardiac dysfunction showing that GRK2 recruitment to the &bgr;AR is G&bgr;&ggr; independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNF&agr;-mediated &bgr;AR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNF&agr;-mediated loss in contractility, showing that TNF&agr;-induced &bgr;AR desensitization is GRK2 dependent. Conclusions— TNF&agr;-induced &bgr;AR desensitization is mediated by GRK2 and is independent of G&bgr;&ggr;, uncovering a hitherto unknown cross-talk between TNF&agr; and &bgr;AR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.

G-Protein Coupled Receptor Resensitization - Appreciating the Balancing Act of Receptor Function

G-protein coupled receptors (GPCRs) are seven transmembrane receptors that are pivotal regulators of cellular responses including vision, cardiac contractility, olfaction, and platelet activation. GPCRs have been a major target for drug discovery due to their role in regulating a broad range of physiological and pathological responses. GPCRs mediate these responses through a cyclical process of receptor activation (initiation of downstream signals), desensitization (inactivation that results in diminution of downstream signals), and resensitization (receptor reactivation for next wave of activation). Although these steps may be of equal importance in regulating receptor function, significant advances have been made in understanding activation and desensitization with limited effort towards resensitization. Inadequate importance has been given to resensitization due to the understanding that resensitization is a homeostasis maintaining process and is not acutely regulated. Evidence indicates that resensitization is a critical step in regulating GPCR function and may contribute towards receptor signaling and cellular responses. In light of these observations, it is imperative to discuss resensitization as a dynamic and mechanistic regulator of GPCR function. In this review we discuss components regulating GPCR function like activation, desensitization, and internalization with special emphasis on resensitization. Although we have used β-adrenergic receptor as a proto-type GPCR to discuss mechanisms regulating receptor function, other GPCRs are also described to put forth a view point on the universality of such mechanisms.

Dominance of the α1B-Adrenergic Receptor and its Subcellular Localization in Human and TRAMP Prostate Cancer Cell Lines

The function and distribution of α1-adrenergic receptor (AR) subtypes in prostate cancer cells is well characterized. Previous studies have used RNA localization or low-avidity antibodies in tissue or cell lines to determine the α1-AR subtype and suggested that the α1 A-AR is dominant. Two androgen-insensitive, human metastatic cancer cell lines DU145 and PC3 were used as well as the mouse TRAMP C1-C3 primary and clonal cell lines. The density of α1-ARs was determined by saturation binding and the distribution of the different α1-AR subtypes was examined by competition-binding experiments. In contrast to previous studies, the major α1-AR subtype in DU145, PC3 and all of the TRAMP cell lines is the α1B-AR. DU145 cells contained 100% of the α1B-AR subtype, whereas PC3 cells were composed of 21% α1 A-AR and 79% α1B-AR. TRAMP cell lines contained between 66% and 79% of the α1B-AR with minor fractions of the other two subtypes. Faster doubling time in the TRAMP cell lines correlated with decreasing α 1B-AR and increasing α1 A- and α1D-AR densities. Transfection with EGFP-tagged α1B-ARs revealed that localization was mainly intracellular, but the majority of the receptors translocated to the cell surface after extended preincubation (18 hr) with either agonist or antagonist. Localization was confirmed by ligand-binding studies and inositol phosphate assays where prolonged preincubation with either agonist and/or antagonist increased the density and function of α 1-ARs, suggesting that the native receptors were mostly intracellular and nonfunctional. Our studies indicate that α1B-ARs are the major α1-AR subtype expressed in DU145, PC3, and all TRAMP cell lines, but most of the receptor is localized in intracellular compartments in a nonfunctional state, which can be rescued upon prolonged incubation with any ligand.

Phosphorylation inactivation of endothelial nitric oxide synthesis in pulmonary arterial hypertension.

The impairment of vasodilator nitric oxide (NO) production is well accepted as a typical marker of endothelial dysfunction in vascular diseases, including in the pathophysiology of pulmonary arterial hypertension (PAH), but the molecular mechanisms accounting for loss of NO production are unknown. We hypothesized that low NO production by pulmonary arterial endothelial cells in PAH is due to inactivation of NO synthase (eNOS) by aberrant phosphorylation of the protein. To test the hypothesis, we evaluated eNOS levels, dimerization, and phosphorylation in the vascular endothelial cells and lungs of patients with PAH compared with controls. In mechanistic studies, eNOS activity in endothelial cells in PAH lungs was found to be inhibited due to phosphorylation at T495. Evidence pointed to greater phosphorylation/activation of protein kinase C (PKC) α and its greater association with eNOS as the source of greater phosphorylation at T495. The presence of greater amounts of pT495-eNOS in plexiform lesions in lungs of patients with PAH confirmed the pathobiological mechanism in vivo. Transfection of the activating mutation of eNOS (T495A/S1177D) restored NO production in PAH cells. Pharmacological blockade of PKC activity by β-blocker also restored NO formation by PAH cells, identifying one mechanism by which β-blockers may benefit PAH and cardiovascular diseases through recovery of endothelial functions.

Phosphoinositide 3-kinase γ inhibits cardiac GSK-3 independently of Akt

A kinase promotes cardiac hypertrophy through a kinase-independent mechanism. Making a Bigger Heart Pathological cardiac hypertrophy can be fatal because it can cause congestive heart failure and arrhythmias. Glycogen synthase kinase-3 (GSK-3), which inhibits cardiac hypertrophy, is active when dephosphorylated by the protein phosphatase PP2A, the activity of which is stimulated by methylation mediated by the methyltransferase PPMT-1. Mohan et al. found that mice lacking the γ isoform of phosphoinositide 3-kinase (PI3K) had smaller hearts than wild-type mice and showed decreased phosphorylation of GSK-3. In addition, these mice showed increased activity of PP2A and PPMT-1. Biochemical experiments indicated that PI3Kγ inhibited the interaction between PP2A and PPMT-1. Heart size and phosphorylation of GSK-3 were increased, and the association of PP2A with PPMT-1 was decreased in PI3Kγ knockout mice by expression of a catalytically inactive form of PI3Kγ. Thus, PI3Kγ promotes cardiac hypertrophy by attenuating the PP2A–PPMT-1 interaction and the inactivation of GSK-3 in a kinase-independent manner. Activation of cardiac phosphoinositide 3-kinase α (PI3Kα) by growth factors, such as insulin, or activation of PI3Kγ downstream of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors stimulates the activity of the kinase Akt, which phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). We found that PI3Kγ inhibited GSK-3 independently of the insulin-PI3Kα-Akt axis. Although insulin treatment activated Akt in PI3Kγ knockout mice, phosphorylation of GSK-3 was decreased compared to control mice. GSK-3 is activated when dephosphorylated by the protein phosphatase 2A (PP2A), which is activated when methylated by the PP2A methyltransferase PPMT-1. PI3Kγ knockout mice showed increased activity of PPMT-1 and PP2A and enhanced nuclear export of the GSK-3 substrate NFATc3. GSK-3 inhibits cardiac hypertrophy, and the hearts of PI3Kγ knockout mice were smaller compared to those of wild-type mice. Cardiac overexpression of a catalytically inactive PI3Kγ (PI3Kγinact) transgene in PI3Kγ knockout mice reduced the activities of PPMT-1 and PP2A and increased phosphorylation of GSK-3. Furthermore, PI3Kγ knockout mice expressing the PI3Kγinact transgene had larger hearts than wild-type or PI3Kγ knockout mice. Our studies show that a kinase-independent function of PI3Kγ could directly inhibit GSK-3 function by preventing the PP2A–PPMT-1 interaction and that this inhibition of GSK-3 was independent of Akt.