Federico Damilano

Integrating Cardiac PIP3 and cAMP Signaling through a PKA Anchoring Function of p110γ

Adrenergic stimulation of the heart engages cAMP and phosphoinositide second messenger signaling cascades. Cardiac phosphoinositide 3-kinase p110γ participates in these processes by sustaining β-adrenergic receptor internalization through its catalytic function and by controlling phosphodiesterase 3B (PDE3B) activity via an unknown kinase-independent mechanism. We have discovered that p110γ anchors protein kinase A (PKA) through a site in its N-terminal region. Anchored PKA activates PDE3B to enhance cAMP degradation and phosphorylates p110γ to inhibit PIP(3) production. This provides local feedback control of PIP(3) and cAMP signaling events. In congestive heart failure, p110γ is upregulated and escapes PKA-mediated inhibition, contributing to a reduction in β-adrenergic receptor density. Pharmacological inhibition of p110γ normalizes β-adrenergic receptor density and improves contractility in failing hearts.

PI3K inhibition in inflammation: Toward tailored therapies for specific diseases

In the past decade, the availability of genetically modified animals has enabled the discovery of interesting roles for phosphatidylinositol 3‐kinase‐γ (PI3Kγ) and ‐δ (PI3Kδ) in different cell types orchestrating innate and adaptive immune responses. Therefore, these PI3K isoforms appear to be attractive drug targets for the treatment of diseases caused by unrestrained immune reactions. Currently, pharmacological targeting of PI3Kγ and/or PI3Kδ represents one of the most promising challenges for companies interested in the development of novel safe treatments for inflammatory diseases. In this review we provide a general outline of PI3Kγ‐ and PI3Kδ‐specific functions in distinct subsets of inflammatory cells. We also discuss the therapeutic impact of novel compounds targeting PI3Kγ, PI3Kδ or both, in mouse models of autoimmune disorders (systemic lupus erythematosus (SLE) and rheumatoid arthritis), respiratory diseases (allergic asthma and chronic obstructive pulmonary disease) and cardiovascular dysfunctions (atherosclerosis and myocardial infarction).

Phosphoinositide 3-Kinase γ Gene Knockout Impairs Postischemic Neovascularization and Endothelial Progenitor Cell Functions

Objective—We evaluated whether phosphatidylinositol 3-kinase γ (PI3Kγ) plays a role in reparative neovascularization and endothelial progenitor cell (EPC) function. Methods and Results—Unilateral limb ischemia was induced in mice lacking the PI3Kγ gene (PI3Kγ−/−) or expressing a catalytically inactive mutant (PI3KγKD/KD) and wild-type controls (WT). Capillarization and arteriogenesis were reduced in PI3Kγ−/− ischemic muscles resulting in delayed reperfusion compared with WT, whereas reparative neovascularization was preserved in PI3KγKD/KD. In PI3Kγ−/− muscles, endothelial cell proliferation was reduced, apoptosis was increased, and interstitial space was infiltrated with leukocytes but lacked cKit+ progenitor cells that in WT muscles typically surrounded arterioles. PI3Kγ is constitutively expressed by WT EPCs, with expression levels being upregulated by hypoxia. PI3Kγ−/− EPCs showed a defect in proliferation, survival, integration into endothelial networks, and migration toward SDF-1. The dysfunctional phenotype was associated with nuclear constraining of FOXO1, reduced Akt and eNOS phosphorylation, and decreased nitric oxide (NO) production. Pretreatment with an NO donor corrected the migratory defect of PI3Kγ−/− EPCs. PI3KγKD/KD EPCs showed reduced Akt phosphorylation, but constitutive activation of eNOS and preserved proliferation, survival, and migration. Conclusions—We newly demonstrated that PI3Kγ modulates angiogenesis, arteriogenesis, and vasculogenesis by mechanisms independent from its kinase activity.

Involvement of Phosphoinositide 3-Kinase γ in Angiogenesis and Healing of Experimental Myocardial Infarction in Mice

Rationale: Phosphoinositide 3-kinase (PI3K)&ggr; is expressed in hematopoietic cells, endothelial cells (ECs), and cardiomyocytes and regulates different cellular functions relevant to inflammation, tissue remodeling and cicatrization. Recently, PI3K&ggr; inhibitors have been indicated for the treatment of chronic inflammatory/autoimmune diseases and atherosclerosis. Objective: We aimed to determine PI3K&ggr; contribution to the angiogenic capacity of ECs and the effect of PI3K&ggr; inhibition on healing of myocardial infarction (MI). Methods and Results: Human umbilical ECs were treated with a selective PI3K&ggr; inhibitor, AS605240, or a pan-phosphoinositide 3-kinases inhibitor, LY294002. Both inhibitory treatments and small interfering RNA-mediated PI3K&ggr; knockdown strongly impaired ECs angiogenic capacity, because of suppression of the PI3K/Akt and mitogen-activated protein kinase pathways. Constitutive activation of Akt rescued the angiogenic defect. Reparative angiogenesis was studied in vivo in a model of MI. AS605240 did not affect MI-induced PI3K&ggr; upregulation, whereas it suppressed Akt activation and downstream signaling. AS605240 strongly reduced inflammation, enhanced cardiomyocyte apoptosis, and impaired survival and proliferation of ECs in peri-infarct zone, which resulted in defective reparative neovascularization. As a consequence, AS605240-treated MI hearts showed increased infarct size and impaired recovery of left ventricular function. Similarly, PI3K&ggr;-deficient mice showed impaired reparative neovascularization, enhanced cardiomyocyte apoptosis and marked deterioration of cardiac function following MI. Mice expressing catalytically inactive PI3K&ggr; also failed to mount a proper neovascularization, although cardiac dysfunction was similar to wild-type controls. Conclusions: PI3K&ggr; expression and catalytic activity are involved at different levels in reparative neovascularization and healing of MI.

Loss of PI3Kγ Enhances cAMP-Dependent MMP Remodeling of the Myocardial N-Cadherin Adhesion Complexes and Extracellular Matrix in Response to Early Biomechanical Stress

Rationale: Mechanotransduction and the response to biomechanical stress is a fundamental response in heart disease. Loss of phosphoinositide 3-kinase (PI3K)&ggr;, the isoform linked to G protein–coupled receptor signaling, results in increased myocardial contractility, but the response to pressure overload is controversial. Objective: To characterize molecular and cellular responses of the PI3K&ggr; knockout (KO) mice to biomechanical stress. Methods and Results: In response to pressure overload, PI3K&ggr;KO mice deteriorated at an accelerated rate compared with wild-type mice despite increased basal myocardial contractility. These functional responses were associated with compromised phosphorylation of Akt and GSK-3&agr;. In contrast, isolated single cardiomyocytes from banded PI3K&ggr;KO mice maintained their hypercontractility, suggesting compromised interaction with the extracellular matrix as the primary defect in the banded PI3K&ggr;KO mice. &bgr;-Adrenergic stimulation increased cAMP levels with increased phosphorylation of CREB, leading to increased expression of cAMP-responsive matrix metalloproteinases (MMPs), MMP2, MT1-MMP, and MMP13 in cardiomyocytes and cardiofibroblasts. Loss of PI3K&ggr; resulted in increased cAMP levels with increased expression of MMP2, MT1-MMP, and MMP13 and increased MMP2 activation and collagenase activity in response to biomechanical stress. Selective loss of N-cadherin from the adhesion complexes in the PI3K&ggr;KO mice resulted in reduced cell adhesion. The &bgr;-blocker propranolol prevented the upregulation of MMPs, whereas MMP inhibition prevented the adverse remodeling with both therapies, preventing the functional deterioration in banded PI3K&ggr;KO mice. In banded wild-type mice, long-term propranolol prevented the adverse remodeling and systolic dysfunction with preservation of the N-cadherin levels. Conclusions: The enhanced propensity to develop heart failure in the PI3K&ggr;KO mice is attributable to a cAMP-dependent upregulation of MMP expression and activity and disorganization of the N-cadherin/&bgr;-catenin cell adhesion complex. &bgr;-Blocker therapy prevents these changes thereby providing a novel mechanism of action for these drugs.

Adipose tissue mitochondrial dysfunction triggers a lipodystrophic syndrome with insulin resistance, hepatosteatosis, and cardiovascular complications

Mitochondrial dysfunction in adipose tissue occurs in obesity, type 2 diabetes, and some forms of lipodystrophy, but whether this dysfunction contributes to or is the result of these disorders is unknown. To investigate the physiological consequences of severe mitochondrial impairment in adipose tissue, we generated mice deficient in mitochondrial transcription factor A (TFAM) in adipocytes by using mice carrying adiponectin‐Cre and TFAM floxed alleles. These adiponectin TFAM‐knockout (adipo‐TFAM‐KO) mice had a 75–81% reduction in TFAM in the subcutaneous and intra‐abdominal white adipose tissue (WAT) and interscapular brown adipose tissue (BAT), causing decreased expression and enzymatic activity of proteins in complexes I, III, and IV of the electron transport chain (ETC). This mitochondrial dysfunction led to adipocyte death and inflammation in WAT and a whitening of BAT. As a result, adipo‐TFAM‐KO mice were resistant to weight gain, but exhibited insulin resistance on both normal chow and high‐fat diets. These lipodystrophic mice also developed hypertension, cardiac hypertrophy, and cardiac dysfunction. Thus, isolated mitochondrial dysfunction in adipose tissue can lead a syndrome of lipodystrophy with metabolic syndrome and cardiovascular complications.—Vernochet, C., Damilano, F., Mourier, A., Bezy, O., Mori, M. A., Smyth, G., Rosenzweig, A., Larsson, N.‐G., Kahn, C. R. Adipose tissue mitochondrial dysfunction triggers a lipodystrophic syndrome with insulin resistance, hepatosteatosis, and cardiovascular complications. FASEB J. 28, 4408–4419 (2014). www.fasebj.org

Phosphoinositide 3-Kinase γ Protects Against Catecholamine-Induced Ventricular Arrhythmia Through Protein Kinase A–Mediated Regulation of Distinct Phosphodiesterases

Background— Phosphoinositide 3-kinase &ggr; (PI3K&ggr;) signaling engaged by &bgr;-adrenergic receptors is pivotal in the regulation of myocardial contractility and remodeling. However, the role of PI3K&ggr; in catecholamine-induced arrhythmia is currently unknown. Methods and Results— Mice lacking PI3K&ggr; (PI3K&ggr;−/−) showed runs of premature ventricular contractions on adrenergic stimulation that could be rescued by a selective &bgr;2-adrenergic receptor blocker and developed sustained ventricular tachycardia after transverse aortic constriction. Consistently, fluorescence resonance energy transfer probes revealed abnormal cAMP accumulation after &bgr;2-adrenergic receptor activation in PI3K&ggr;−/− cardiomyocytes that depended on the loss of the scaffold but not of the catalytic activity of PI3K&ggr;. Downstream from &bgr;-adrenergic receptors, PI3K&ggr; was found to participate in multiprotein complexes linking protein kinase A to the activation of phosphodiesterase (PDE) 3A, PDE4A, and PDE4B but not of PDE4D. These PI3K&ggr;-regulated PDEs lowered cAMP and limited protein kinase A–mediated phosphorylation of L-type calcium channel (Cav1.2) and phospholamban. In PI3K&ggr;−/− cardiomyocytes, Cav1.2 and phospholamban were hyperphosphorylated, leading to increased Ca2+ spark occurrence and amplitude on adrenergic stimulation. Furthermore, PI3K&ggr;−/− cardiomyocytes showed spontaneous Ca2+ release events and developed arrhythmic calcium transients. Conclusions— PI3K&ggr; coordinates the coincident signaling of the major cardiac PDE3 and PDE4 isoforms, thus orchestrating a feedback loop that prevents calcium-dependent ventricular arrhythmia.

Distinct Effects of Leukocyte and Cardiac Phosphoinositide 3-Kinase γ Activity in Pressure Overload–Induced Cardiac Failure

Background— Signaling from phosphoinositide 3-kinase &ggr; (PI3K&ggr;) is crucial for leukocyte recruitment and inflammation but also contributes to cardiac maladaptive remodeling. To better understand the translational potential of these findings, this study investigates the role of PI3K&ggr; activity in pressure overload–induced heart failure, addressing the distinct contributions of bone marrow–derived and cardiac cells. Methods and Results— After transverse aortic constriction, mice knock-in for a catalytically inactive PI3K&ggr; (PI3K&ggr; KD) showed reduced fibrosis and normalized cardiac function up to 16 weeks. Accordingly, treatment with a selective PI3K&ggr; inhibitor prevented transverse aortic constriction–induced fibrosis. To define the cell types involved in this protection, bone marrow chimeras, lacking kinase activity in the immune system or the heart, were studied after transverse aortic constriction. Bone marrow–derived cells from PI3K&ggr; KD mice were not recruited to wild-type hearts, thus preventing fibrosis and preserving diastolic function. After prolonged pressure overload, chimeras with PI3K&ggr; KD bone marrow–derived cells showed slower development of left ventricular dilation and higher fractional shortening than controls. Conversely, in the presence of a wild-type immune system, KD hearts displayed bone marrow–derived cell infiltration and fibrosis at early stages but reduced left ventricular dilation and preserved contractile function at later time points. Conclusions— Together, these data demonstrate that, in response to transverse aortic constriction, PI3K&ggr; contributes to maladaptive remodeling at multiple levels by modulating both cardiac and immune cell functions.

PI3Kγ Protects against Catecholamine-Induced Ventricular Arrhythmia through PKA-mediated Regulation of Distinct Phosphodiesterases

Background— Phosphoinositide 3-kinase &ggr; (PI3K&ggr;) signaling engaged by &bgr;-adrenergic receptors is pivotal in the regulation of myocardial contractility and remodeling. However, the role of PI3K&ggr; in catecholamine-induced arrhythmia is currently unknown. Methods and Results— Mice lacking PI3K&ggr; (PI3K&ggr;−/−) showed runs of premature ventricular contractions on adrenergic stimulation that could be rescued by a selective &bgr;2-adrenergic receptor blocker and developed sustained ventricular tachycardia after transverse aortic constriction. Consistently, fluorescence resonance energy transfer probes revealed abnormal cAMP accumulation after &bgr;2-adrenergic receptor activation in PI3K&ggr;−/− cardiomyocytes that depended on the loss of the scaffold but not of the catalytic activity of PI3K&ggr;. Downstream from &bgr;-adrenergic receptors, PI3K&ggr; was found to participate in multiprotein complexes linking protein kinase A to the activation of phosphodiesterase (PDE) 3A, PDE4A, and PDE4B but not of PDE4D. These PI3K&ggr;-regulated PDEs lowered cAMP and limited protein kinase A–mediated phosphorylation of L-type calcium channel (Cav1.2) and phospholamban. In PI3K&ggr;−/− cardiomyocytes, Cav1.2 and phospholamban were hyperphosphorylated, leading to increased Ca2+ spark occurrence and amplitude on adrenergic stimulation. Furthermore, PI3K&ggr;−/− cardiomyocytes showed spontaneous Ca2+ release events and developed arrhythmic calcium transients. Conclusions— PI3K&ggr; coordinates the coincident signaling of the major cardiac PDE3 and PDE4 isoforms, thus orchestrating a feedback loop that prevents calcium-dependent ventricular arrhythmia.

PI3K kinase and scaffold functions in heart

Signal transduction events are key modulators of cellular function and, in the cardiovascular system, an emerging role is played by phosphoinositide 3‐kinases (PI3Ks), a family of enzymes containing a 3‐phosphorylated phosphoinositide that produce lipid second messengers. In the heart, multiple PI3K isoforms are expressed, but play potentially distinct roles. Among cardiac PI3Ks, PI3Kα is triggered by tyrosine kinase receptors and plays a role in adaptive hypertrophy, while PI3Kγ is triggered by G protein–coupled receptors and is involved in maladaptive heart remodeling. This view has been recently complicated by the finding that PI3Ks can also be involved in protein–protein interactions and affect signaling independently of their kinase activity. This review will thus focus on the effects of these multiple signaling events, with particular emphasis on their involvement in cardiac hypertrophy and failure.