Valerie Myers

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the “paradoxical” Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.

Controlled and Cardiac-Restricted Overexpression of the Arginine Vasopressin V1A Receptor Causes Reversible Left Ventricular Dysfunction Through Gαq-Mediated Cell Signaling

BACKGROUND [Arg8]-vasopressin (AVP) activates 3 G-protein-coupled receptors: V1A, V2, and V1B. The AVP-V1A receptor is the primary AVP receptor in the heart; however, its role in cardiac homeostasis is controversial. To better understand AVP-mediated signaling in the heart, we created a transgenic mouse with controlled overexpression of the V1A receptor. METHODS AND RESULTS The V1A receptor transgene was placed under the control of the tetracycline-regulated, cardiac-specific α-myosin heavy chain promoter (V1A-TG). V1A-TG mice had a normal cardiac function phenotype at 10 weeks of age; however, by 24 weeks of age, tetracycline-transactivating factor/V1A-TG mouse hearts had reduced cardiac function, cardiac hypertrophy, and dilatation of the ventricular cavity. Contractile dysfunction was also observed in isolated adult cardiac myocytes. When V1A receptor transgene was induced to be expressed in adult mice (V1A-TG(Ind)), left ventricular dysfunction and dilatation were also seen, albeit at a later time point. Because the V1A receptor mediates cell signaling through Gα(q) protein, we blocked Gα(q) signaling by crossing tetracycline-transactivating factor/V1A mice with transgenic mice that expressed a small inhibitory peptide against Gα(q). Gα(q) blockade abrogated the development of the heart failure phenotype in tetracycline-transactivating factor/V1A-TG mice. The heart failure phenotype could be reversed by administration of doxycycline. CONCLUSIONS Our results demonstrate a role for V1A-mediated signaling in the development of heart failure and support a role for V1A blockade in the treatment of patients with elevated levels of vasopressin.

Myocardial injury after ischemia-reperfusion in mice deficient in Akt2 is associated with increased cardiac macrophage density.

Akt2 protein kinase has been shown to promote cell migration and actin polymerization in several cell types, including macrophages. Because migrating macrophages constitute an important inflammatory response after myocardial ischemia, we determined cardiac macrophage expression after ischemia-reperfusion (I/R) injury and cryo-injury in mice lacking Akt2 (Akt2-KO). At 7 days post-I/R, Akt2-KO cardiac tissues showed an increase in immunohistochemical staining for macrophage markers (Galectin 3 and F4/80) compared with wild-type (WT) mice, indicating macrophage density was increased in the injured Akt2-KO myocardium. This change was time dependent because macrophage density was similar between WT and Akt2-KO myocardium at 3 days post-I/R, but by 7 and 14 days post-I/R, macrophage density was significantly increased in Akt2-KO myocardium. Concomitantly, infarct size was larger and cardiac function was reduced in Akt2-KO mice subjected to I/R. However, when cryo-infarction produced similar infarct sizes in the anterior wall in both WT and Akt2-KO mice, macrophage density remained higher in Akt2-KO mouse myocardium, suggesting Akt2 regulates myocardial macrophage density independent of infarct size. Consistently, bone marrow from Akt2-KO mice enhanced myocardial macrophage density in both C57/B6 WT and Akt2-KO recipient mice. Finally, reciprocal ex-vivo coculturing of macrophages and cardiac myocytes showed that activated Akt2-KO peritoneal macrophages had reduced mobility and adhesion when compared with WT littermate controls. Thus, although Akt-2 KO mice did not affect the initial inflammation response after injury and Akt2 deficiency has been shown to impair cell migration or motility in macrophages, our data suggested a novel mechanism in which increasing retention of Akt2-KO macrophages resulted in increasing cardiac Akt2-KO macrophage density in the myocardial space.

Bcl-2–associated athanogene 3 protects the heart from ischemia/reperfusion injury

Bcl-2-associated athanogene 3 (BAG3) is an evolutionarily conserved protein expressed at high levels in the heart and the vasculature and in many cancers. While altered BAG3 expression has been associated with cardiac dysfunction, its role in ischemia/reperfusion (I/R) is unknown. To test the hypothesis that BAG3 protects the heart from reperfusion injury, in vivo cardiac function was measured in hearts infected with either recombinant adeno-associated virus serotype 9-expressing (rAAV9-expressing) BAG3 or GFP and subjected to I/R. To elucidate molecular mechanisms by which BAG3 protects against I/R injury, neonatal mouse ventricular cardiomyocytes (NMVCs) in which BAG3 levels were modified by adenovirus expressing (Ad-expressing) BAG3 or siBAG3 were exposed to hypoxia/reoxygenation (H/R). H/R significantly reduced NMVC BAG3 levels, which were associated with enhanced expression of apoptosis markers, decreased expression of autophagy markers, and reduced autophagy flux. The deleterious effects of H/R on apoptosis and autophagy were recapitulated by knockdown of BAG3 with Ad-siBAG3 and were rescued by Ad-BAG3. In vivo, treatment of mice with rAAV9-BAG3 prior to I/R significantly decreased infarct size and improved left ventricular function when compared with mice receiving rAAV9-GFP and improved markers of autophagy and apoptosis. These findings suggest that BAG3 may provide a therapeutic target in patients undergoing reperfusion after myocardial infarction.

Effects of cardiac-restricted overexpression of the A2A adenosine receptor on adriamycin-induced cardiotoxicity

Activation of the A(2A) adenosine receptor (A(2A)R) has been shown to be cardioprotective. We hypothesized that A(2A)R overexpression could protect the heart from adriamycin-induced cardiomyopathy. Transgenic (TG) mice overexpressing the A(2A)R and wild-type mice (WT) were injected with adriamycin (5 mg.kg(-1).wk(-1) ip, 4 wk). All WT mice survived adriamycin treatment while A(2A)R TG mice suffered 100% mortality at 4 wk. Telemetry showed progressive prolongation of the QT interval, bradyarrhythmias, heart block, and sudden death in adriamycin-treated A(2A)R TG but not WT mice. Both WT and A(2A)R TG demonstrated similar decreases in heart function at 3 wk after treatment. Adriamycin significantly increased end-diastolic intracellular Ca(2+) concentration in A(2A)R TG but not in WT myocytes (P < 0.05). Compared with WT myocytes, action potential duration increased dramatically in A(2A)R TG myocytes (P < 0.05) after adriamycin treatment. Expression of connexin 43 was decreased in adriamycin treated A(2A)R TG but not WT mice. In sharp contrast, A(2A)R overexpression induced after the completion of adriamycin treatment resulted in no deaths and enhanced cardiac performance compared with WT adriamycin-treated mice. Our results indicate that the timing of A(2A)R activation is critical in terms of exacerbating or protecting adriamycin-induced cardiotoxicity. Our data have direct relevance on the clinical use of adenosine agonists or antagonists in the treatment of patients undergoing adriamycin therapy.

Haplo-insufficiency of Bcl2-associated athanogene 3 in mice results in progressive left ventricular dysfunction, β-adrenergic insensitivity, and increased apoptosis

Bcl2‐associated athanogene 3 (BAG3) is a 575 amino acid protein that is found predominantly in the heart, skeletal muscle, and many cancers. Deletions and truncations in BAG3 that result in haplo‐insufficiency have been associated with the development of dilated cardiomyopathy. To study the cellular and molecular events attributable to BAG3 haplo‐insufficiency we generated a mouse in which one allele of BAG3 was flanked by loxP recombination sites (BAG3fl/+). Mice were crossed with α‐MHC‐Cre mice in order to generate mice with cardiac‐specific haplo‐insufficiency (cBAG3+/−) and underwent bi–weekly echocardiography to assess their cardiac phenotype. By 10 weeks of age, cBAG3+/− mice demonstrated increased heart size and diminished left ventricular ejection fraction when compared with non‐transgenic littermates (Cre−/−BAG3fl/+). Contractility in adult myocytes isolated from cBAG3+/− mice were similar to those isolated from control mice at baseline, but showed a significantly decreased response to adrenergic stimulation. Intracellular calcium ([Ca2+]i) transient amplitudes in myocytes isolated from cBAG3+/− mice were also similar to myocytes isolated from control mice at baseline but were significantly lower than myocytes from control mice in their response to isoproterenol. BAG3 haplo‐insufficiency was also associated with decreased autophagy flux and increased apoptosis. Taken together, these results suggest that mice in which BAG3 has been deleted from a single allele provide a model that mirrors the biology seen in patients with heart failure and BAG3 haplo‐insufficiency.

The Multifunctional Protein BAG3: A Novel Therapeutic Target in Cardiovascular Disease

Summary The B-cell lymphoma 2–associated anthanogene (BAG3) protein is expressed most prominently in the heart, the skeletal muscle, and in many forms of cancer. In the heart, it serves as a co-chaperone with heat shock proteins in facilitating autophagy; binds to B-cell lymphoma 2, resulting in inhibition of apoptosis; attaches actin to the Z disk, providing structural support for the sarcomere; and links the α-adrenergic receptor with the L-type Ca2+ channel. When BAG3 is overexpressed in cancer cells, it facilitates prosurvival pathways that lead to insensitivity to chemotherapy, metastasis, cell migration, and invasiveness. In contrast, in the heart, mutations in BAG3 have been associated with a variety of phenotypes, including both hypertrophic/restrictive and dilated cardiomyopathy. In murine skeletal muscle and vasculature, a mutation in BAG3 leads to critical limb ischemia after femoral artery ligation. An understanding of the biology of BAG3 is relevant because it may provide a therapeutic target in patients with both cardiac and skeletal muscle disease.

β-Adrenergic Receptor–Mediated Cardiac Contractility Is Inhibited via Vasopressin Type 1A-Receptor–Dependent SignalingCLINICAL PERSPECTIVE

Background— Enhanced arginine vasopressin levels are associated with increased mortality during end-stage human heart failure, and cardiac arginine vasopressin type 1A receptor (V1AR) expression becomes increased. Additionally, mice with cardiac-restricted V1AR overexpression develop cardiomyopathy and decreased &bgr;-adrenergic receptor (&bgr;AR) responsiveness. This led us to hypothesize that V1AR signaling regulates &bgr;AR responsiveness and in doing so contributes to development of heart failure. Methods and Results— Transaortic constriction resulted in decreased cardiac function and &bgr;AR density and increased cardiac V1AR expression, effects reversed by a V1AR-selective antagonist. Molecularly, V1AR stimulation led to decreased &bgr;AR ligand affinity, as well as &bgr;AR-induced Ca2+ mobilization and cAMP generation in isolated adult cardiomyocytes, effects recapitulated via ex vivo Langendorff analysis. V1AR-mediated regulation of &bgr;AR responsiveness was demonstrated to occur in a previously unrecognized Gq protein–independent/G protein receptor kinase–dependent manner. Conclusions— This newly discovered relationship between cardiac V1AR and &bgr;AR may be informative for the treatment of patients with acute decompensated heart failure and elevated arginine vasopressin.