Transgenic Gαq overexpression induces cardiac contractile failure in mice

The critical cell signals that trigger cardiac hypertrophy and regulate the transition to heart failure are not known. To determine the role of Gαq-mediated signaling pathways in these events, transgenic mice were constructed that overexpressed wild-type Gαq in the heart using the α-myosin heavy chain promoter. Two-fold overexpression of Gαq showed no detectable effects, whereas 4-fold overexpression resulted in increased heart weight and myocyte size along with marked increases in atrial naturietic factor (≈55-fold), β-myosin heavy chain (≈8-fold), and α-skeletal actin (≈8-fold) expression, and decreased (≈3-fold) β-adrenergic receptor-stimulated adenylyl cyclase activity. All of these signals have been considered markers of hypertrophy or failure in other experimental systems or human heart failure. Echocardiography and in vivo cardiac hemodynamic studies indeed revealed impaired intrinsic contractility manifested as decreased fractional shortening (19 ± 2% vs. 41 ± 3%), dP/dt max, a negative force–frequency response, an altered Starling relationship, and blunted contractile responses to the β-adrenergic agonist dobutamine. At higher levels of Gαq overexpression, frank cardiac decompensation occurred in 3 of 6 animals with development of biventricular failure, pulmonary congestion, and death. The element within the pathway that appeared to be critical for these events was activation of protein kinase C. Interestingly, mitogen-activated protein kinase, which is postulated by some to be important in the hypertrophy program, was not activated. The Gαq overexpressor exhibits a biochemical and physiologic phenotype resembling both the compensated and decompensated phases of human cardiac hypertrophy and suggests a common mechanism for their pathogenesis.     

Transient cardiac expression of constitutively active Gαq leads to hypertrophy and dilated cardiomyopathy by calcineurin-dependent and independent pathways

Cardiac hypertrophy and dilatation can result from stimulation of signal transduction pathways mediated by heterotrimeric G proteins, especially G_q, whose α subunit activates phospholipase Cβ (PLCβ). We now report that transient, modest expression of a hemagglutinin (HA) epitope-tagged, constitutively active mutant of the G_q α subunit (HAα^*_q) in hearts of transgenic mice is sufficient to induce cardiac hypertrophy and dilatation that continue to progress after the initiating stimulus becomes undetectable. At 2 weeks, HAα^*_q protein is expressed at less than 50% of endogenous α_q/11, and the transgenic hearts are essentially normal morphologically. Although HAα^*_q protein declines at 4 weeks and is undetectable by 10 weeks, the animals develop cardiac hypertrophy and dilatation and die between 8 and 30 weeks in heart failure. As the pathology develops, endogenous α_q/11 rises (2.9-fold in atria; 1.8-fold in ventricles). At 2 weeks, basal PLC activity is increased 9- to 10-fold in atria but not ventricles. By 10 weeks, it is elevated in both, presumably because of the rise in endogenous α_q/11. We conclude that the pathological changes initiated by early, transient HAα^*_q expression are maintained in part by compensatory changes in signal transduction and other pathways. Cyclosporin A (CsA) prevents hypertrophy caused by activation of calcineurin [Molkentin, J. D., Lu, J.-R., Antos, C. L., Markham, B., Richardson, J., Robbins, J., Grant, S. R. & Olson, E. N. (1998) Cell 93, 215–228]. Because HAα^*_q acts upstream of calcineurin, we hypothesized that HAα^*_q might initiate additional pathways leading to hypertrophy and dilatation. Treating HAα^*_q mice with CsA diminished some, but not all, aspects of the hypertrophic phenotype, suggesting that multiple pathways are involved.     

Crosstalk between Gαi- and Gαq-coupled receptors is mediated by Gβγ exchange

Activation of Galpha(i)-coupled receptors often causes enhancement of the inositol phosphate signal triggered by Galpha(q)-coupled receptors. To investigate the mechanism of this synergistic receptor crosstalk, we studied the Galpha(i)-coupled adenosine A(1) and alpha(2C) adrenergic receptors and the Galpha(q)-coupled bradykinin B(2) and a UTP-preferring P2Y receptor. Stimulation of either Galpha(i)-coupled receptor expressed in COS cells increased the potency and the efficacy of inositol phosphate production by bradykinin or UTP. Likewise, overexpression of Gbeta(1)gamma(2) resulted in a similar increase in potency and efficacy of bradykinin or UTP. In contrast, these stimuli did not affect the potency of direct activators of Galpha(q); a truncated Gbeta(3) mutant had no effect on the receptor-generated signals whereas signals generated at the G-protein level were still enhanced. This suggests that the Gbetagamma-mediated signal enhancement occurs at the receptor level. Almost all possible combinations of Gbeta(1-3) with Ggamma(2-7) were equally effective in enhancing the signals of the B(2) and a UTP-preferring P2Y receptor, indicating a very broad specificity of this synergism. The enhancement of the bradykinin signal by (i) Galpha(i)-activating receptor ligands or (ii) cotransfection of Gbetagamma was suppressed when the B(2) receptor was replaced by a B(2)Gbeta(2) fusion protein. Gbetagamma enhanced the B(2) receptor-stimulated activation of G-proteins as determined by GTPgammaS-induced decrease in high affinity agonist binding and by B(2) receptor-enhanced [(35)S]GTPgammaS binding. These findings support the concept that Gbetagamma exchange between Galpha(i)- and Galpha(q)-coupled receptors mediates this type of receptor crosstalk.     

Low- and high-level transgenic expression of β2-adrenergic receptors differentially affect cardiac hypertrophy and function in Gαq-overexpressing mice

Transgenic overexpression of Galphaq in the heart triggers events leading to a phenotype of eccentric hypertrophy, depressed ventricular function, marked expression of hypertrophy-associated genes, and depressed beta-adrenergic receptor (betaAR) function. The role of betaAR dysfunction in the development of this failure phenotype was delineated by transgenic coexpression of the carboxyl terminus of the betaAR kinase (betaARK), which acts to inhibit the kinase, or concomitant overexpression of the beta2AR at low (approximately 30-fold, Galphaq/beta2ARL), moderate (approximately 140-fold, Galphaq/beta2ARM), and high (approximately 1,000-fold, Galphaq/beta2ARH) levels above background betaAR density. Expression of the betaARK inhibitor had no effect on the phenotype, consistent with the lack of increased betaARK levels in Galphaq mice. In marked contrast, Galphaq/beta2ARL mice displayed rescue of hypertrophy and resting ventricular function and decreased cardiac expression of atrial natriuretic factor and alpha-skeletal actin mRNA. These effects occurred in the absence of any improvement in basal or agonist-stimulated adenylyl cyclase (AC) activities in crude cardiac membranes, although restoration of a compartmentalized beta2AR/AC signal cannot be excluded. Higher expression of receptors in Galphaq/beta2ARM mice resulted in salvage of AC activity, but hypertrophy, ventricular function, and expression of fetal genes were unaffected or worsened. With approximately 1,000-fold overexpression, the majority of Galphaq/beta2ARH mice died with cardiomegaly at 5 weeks. Thus, although it appears that excessive, uncontrolled, or generalized augmentation of betaAR signaling is deleterious in heart failure, selective enhancement by overexpressing the beta2AR subtype to limited levels restores not only ventricular function but also reverses cardiac hypertrophy.     

Progressive hypertrophy and heart failure in β1-adrenergic receptor transgenic mice

Stimulation of cardiac beta1-adrenergic receptors is the main mechanism that increases heart rate and contractility. Consequently, several pharmacological and gene transfer strategies for the prevention of heart failure aim at improving the function of the cardiac beta-adrenergic receptor system, whereas current clinical treatment favors a reduction of cardiac stimulation. To address this controversy, we have generated mice with heart-specific overexpression of beta1-adrenergic receptors. Their cardiac function was investigated in organ bath experiments as well as in vivo by cardiac catheterization and by time-resolved NMR imaging. The transgenic mice had increased cardiac contractility at a young age but also developed marked myocyte hypertrophy (3.5-fold increase in myocyte area). This increase was followed by progressive heart failure with functional and histological deficits typical for humans with heart failure. Contractility was reduced by approximately 50% in 35-week-old mice, and ejection fraction was reduced down to a minimum of approximately 20%. We conclude that overexpression of beta1-adrenergic receptors in the heart may lead to a short-lived improvement of cardiac function, but that increased beta1-adrenergic receptor signalling is ultimately detrimental.     

Posttranslational modification of Gαo1 generates Gαo3, an abundant G protein in brain

Gα_o, the most abundant G protein in mammalian brain, occurs at least in two subforms, i.e., Gα_o1 and Gα_o2, derived by alternative splicing of the mRNA. A third Gα_o1-related isoform, Gα_o3, has been purified, representing about 30% of total G_o in brain. Initial studies revealed distinct biochemical properties of Gα_o3 as compared with other Gα_o isoforms. In matrix-assisted laser desorption/ionization peptide mass mapping of gel-isolated Gα_o1 and Gα_o3, C-terminal peptides showed a difference of +1 Da for Gα_o3. Nanoelectrospray tandem mass spectrometry sequencing revealed an Asp instead of an Asn at position 346 of Gα_o3. Gel electrophoretic analysis of recombinant Gα_o3 showed the same mobility as native Gα_o3 but distinct to Gα_o1. The conversion of ³⁴⁶Asn→Asp changed the signaling properties, including the velocity of the basal guanine nucleotide-exchange reaction, which points to the involvement of the C terminus in basal guanosine 5′-[γ-thio]triphosphate binding. No cDNA coding for Gα_o3 was detected, suggesting an enzymatic deamidation of Gα_o1 by a yet-unidentified activity. Therefore, Gα heterogeneity is generated not only at the DNA or RNA levels, but also at the protein level. The relative amount of Gα_o1 and Gα_o3 differed from cell type to cell type, indicating an additional principle of G protein regulation.     

Selective interaction of the C2 domains of phospholipase C-β1 and -β2 with activated Gαq subunits: An alternative function for C2-signaling modules

Phospholipase C (PLC)-beta1 and PLC-beta2 are regulated by the Gq family of heterotrimeric G proteins and contain C2 domains. These domains are Ca2+-binding modules that serve as membrane-attachment motifs in a number of signal transduction proteins. To determine the role that C2 domains play in PLC-beta1 and PLC-beta2 function, we measured the binding of the isolated C2 domains to membrane bilayers. We found, unexpectedly, that these modules do not bind to membranes but they associate strongly and specifically to activated [guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS])-bound] Galphaq subunits. The C2 domain of PLC-beta1 effectively suppressed the activation of the intact isozyme by Galphaq(GTP[gammaS]), indicating that the C2-Galphaq interaction may be physiologically relevant. C2 affinity for Galphaq(GTP[gammaS]) was reduced when Galphaq was deactivated to the GDP-bound state. Binding to activated Galphai1 subunits or to Gbetagamma subunits was not detected. Also, Galphaq(GTP[gammaS]) failed to associate with the C2 domain of PLC-delta, an isozyme that is not activated by Galphaq. These results indicate that the C2 domains of PLC-beta1 and PLC-beta2 provide a surface to which Galphaq subunits can dock, leading to activation of the native protein.     

Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein α subunit Gα13

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein α subunits were characterized in transfection systems. Gαq, Gα12, and Gα13, but not Gαi, activate SRF through RhoA. When Gαq, α12, or α13 were coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, Gα13, but not Gαq or Gα12, showed synergistic activation of SRF with GEF115. The synergy between Gα13 and GEF115 depends on the N-terminal part of GEF115, and there was no synergistic effect between Gα13 and another Rho-specific exchange factor Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibited Gα13- and Gα12-induced, but not GEF115 itself- or Gαq-induced, SRF activation. The DH-domain-deletion mutant also suppressed thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of Gα12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both Gα12 and Gα13. Thus, the inhibition of Gα12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between Gα13 and GEF115 indicates that GEF115 mediates Gα13-induced activation of Rho and SRF.     

Interferon α2–Thymosin α1 Fusion Protein (IFNα2–Tα1): A Genetically Engineered Fusion Protein with Enhanced Anticancer and Antiviral Effect

Human interferon α2 (IFNα2) and thymosin α1 (Tα1) are therapeutic proteins used for the treatment of viral infections and different types of cancer. Both IFNα2 and Tα1 show a synergic effect in their activities when used in combination. Furthermore, the therapeutic fusion proteins produced through the genetic fusion of two genes can exhibit several therapeutic functions in one molecule. In this study, we determined the anticancer and antiviral effect of human interferon α2–thymosin α1 fusion protein (IFNα2–Tα1) produced in our laboratory for the first time. The cytotoxic and genotoxic effect of IFNα2–Tα1 was evaluated in HepG2 and MDA-MB-231 cells. The in vitro assays confirmed that IFNα2–Tα1 inhibited the growth of cells more effectively than IFNα2 alone and showed an elevated genotoxic effect. The expression of proapoptotic genes was also significantly enhanced in IFNα2–Tα1-treated cells compared to IFNα2-treated cells. Furthermore, the HCV RNA level was significantly reduced in IFNα2–Tα1-treated HCV-infected Huh7 cells compared to IFNα2-treated cells. The quantitative PCR analysis showed that the expression of various genes, the products of which inhibit HCV replication, was significantly enhanced in IFNα2–Tα1-treated cells compared to IFNα2-treated cells. Our findings demonstrate that IFNα2–Tα1 is more effective than single IFNα2 as an anticancer and antiviral agent.     

Novel α + β Zr Alloys with Enhanced Strength

Low-alloyed zirconium alloys are widely used in nuclear applications due to their low neutron absorption cross-section. These alloys, however, suffer from limited strength. Well-established guidelines for the development of Ti alloys were applied to design new two-phase ternary Zr alloys with improved mechanical properties. Zr-4Sn-4Nb and Zr-8Sn-4Nb alloys have been manufactured by vacuum arc melting, thermo-mechanically processed by annealing, forging, and aging to various microstructural conditions and thoroughly characterized. Detailed Scanning electron microscopy (SEM) analysis showed that the microstructural response of the alloys is rather similar to alpha + beta Ti alloys. Duplex microstructure containing primary alpha phase particles surrounded by lamellar alpha + beta microstructure can be achieved by thermal processing. Mechanical properties strongly depend on the previous treatment. Ultimate tensile strength exceeding 700 MPa was achieved exceeding the strength of commercial Zr alloys for nuclear applications by more than 50%. Such an improvement in strength more than compensates for the increased neutron absorption cross-section. This study aims to exploit the potential of alpha + beta Zr alloys for nuclear applications.     

Impaired motor coordination and persistent multiple climbing fiber innervation of cerebellar Purkinje cells in mice lacking Gαq

Mice lacking the α-subunit of the heterotrimeric guanine nucleotide binding protein G_q (Gα_q) are viable but suffer from ataxia with typical signs of motor discoordination. The anatomy of the cerebellum is not overtly disturbed, and excitatory synaptic transmission from parallel fibers to cerebellar Purkinje cells (PCs) and from climbing fibers (CFs) to PCs is functional. However, about 40% of adult Gα_q mutant PCs remain multiply innervated by CFs because of a defect in regression of supernumerary CFs in the third postnatal week. Evidence is provided suggesting that Gα_q is part of a signaling pathway that is involved in the elimination of multiple CF innervation during this period.     

Gαo is necessary for muscarinic regulation of Ca2+ channels in mouse heart

Heterotrimeric G proteins, composed of Gα and Gβγ subunits, transmit signals from cell surface receptors to cellular effector enzymes and ion channels. The Gα_o protein is the most abundant Gα subtype in the nervous system, but it is also found in the heart. Its function is not completely known, although it is required for regulation of N-type Ca²⁺ channels in GH₃ cells and also interacts with GAP43, a major protein in growth cones, suggesting a role in neuronal pathfinding. To analyze the function of Gα_o, we have generated mice lacking both isoforms of Gα_o by homologous recombination. Surprisingly, the nervous system is grossly intact, despite the fact that Gα_o makes up 0.2–0.5% of brain particulate protein and 10% of the growth cone membrane. The Gα_o−/− mice do suffer tremors and occasional seizures, but there is no obvious histologic abnormality in the nervous system. In contrast, Gα_o−/− mice have a clear and specific defect in ion channel regulation in the heart. Normal muscarinic regulation of L-type calcium channels in ventricular myocytes is absent in the mutant mice. The L-type calcium channel responds normally to isoproterenol, but there is no evident muscarinic inhibition. Muscarinic regulation of atrial K⁺ channels is normal, as is the electrocardiogram. The levels of other Gα subunits (Gα_s, Gα_q, and Gα_i) are unchanged in the hearts of Gα_o−/− mice, but the amount of Gβγ is decreased. Whichever subunit, Gα_o or Gβγ, carries the signal forward, these studies show that muscarinic inhibition of L-type Ca²⁺ channels requires coupling of the muscarinic receptor to Gα_o. Other cardiac Gα subunits cannot substitute.     

Cardiac adrenomedullin: its role in cardiac hypertrophy and heart failure

Co-localization of adrenomedullin (AM) and its receptor components such as calcitonin receptor like receptor (CRLR), receptor activity modifying protein (RAMP)2 and RAMP3 in peripheral tissues, including the heart, kidney, and vasculature, suggests an important role for the peptide as a regulator of cardiovascular function. Indeed, we previously reported that AM gene expression and / or immunoreactivity are increased in the ventricles of cardiac hypertrophy and heart failure. Recently, we also found that not only levels of AM peptide and AM gene expression, but also mRNA levels of CRLR, RAMP2 and RAMP3 are increased in cardiac hypertrophy and failing heart. Cardiac myocytes and fibroblast produce and secrete two molecular forms of AM and express CRLR, RAMP2 and RAMP3, and AM is known to have inhibitory effect of collagen synthesis and antiproliferative effect in cardiac fibroblasts. Stimulation by IL-1beta significantly increased gene expression of AM and its receptor components in cardiac fibroblasts. Preincubated IL-1beta elevated the intracellular cAMP response to exogenous administered AM. AM antisense oligodeoxynucleotide treatment significantly lowered AM levels in cultured medium. IL-1beta significantly increased (3)H-proline incorporation and AM antisense oligodeoxynucleotide treatment further increased (3)H-proline incorporation. Collectively, these results support a protective role for increased AM in the cardiac hypertrophy and heart failure. Then, we tested the effects of acute administration of AM in experimental and human heart failure, because AM has hemodynamic effects including vasodilation, increases in cardiac contractility, cardiac output, diuresis, and natriuresis. We observed profound and sustained cardiovascular, hormonal and renal effects. These effects may incorporate many of the therapeutic goals of heart failure management.     

Receptor and Gβγ isoform-specific interactions with G protein-coupled receptor kinases

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free βγ subunits of heterotrimeric G proteins (Gβγ). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of Gβγ with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a β-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known Gβγ sequestrant. Furthermore, in cells overexpressing both GRK2 and Gβ₁γ₂, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/Gβγ complex. That Gβγ specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/Gβγ complex is formed after agonist occupancy of the lysophosphatidic acid and β-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a Gβγ complex after stimulation of all three GPCRs. This Gβγ binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds Gβ₁ and Gβ₂ but not Gβ₃, while the GRK3 fusion protein binds all three Gβ isoforms. This study provides a direct demonstration of a role for Gβγ in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.     

Transgenic model of aldosterone-driven cardiac hypertrophy and heart failure

Aldosterone classically promotes unidirectional transepithelial sodium transport, thereby regulating blood volume and blood pressure. Recently, both clinical and experimental studies have suggested additional, direct roles for aldosterone in the cardiovascular system. To evaluate aldosterone activation of cardiomyocyte mineralocorticoid receptors, transgenic mice overexpressing 11beta-hydroxysteroid dehydrogenase type 2 in cardiomyocytes were generated using the mouse alpha-myosin heavy chain promoter. This enzyme converts glucocorticoids to receptor-inactive metabolites, allowing aldosterone occupancy of cardiomyocyte mineralocorticoid receptors. Transgenic mice were normotensive but spontaneously developed cardiac hypertrophy, fibrosis, and heart failure and died prematurely on a normal salt diet. Eplerenone, a selective aldosterone blocker, ameliorated this phenotype. These studies confirm the deleterious consequences of inappropriate activation of cardiomyocyte mineralocorticoid receptors by aldosterone and reveal a tonic inhibitory role of glucocorticoids in preventing such outcomes under physiological conditions. In addition, these data support the hypothesis that aldosterone blockade may provide additional therapeutic benefit in the treatment of heart failure.