Alessandra Ghigo

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).

Hemopexin Therapy Improves Cardiovascular Function by Preventing Heme-Induced Endothelial Toxicity in Mouse Models of Hemolytic Diseases

Background— Hemolytic diseases are characterized by enhanced intravascular hemolysis resulting in heme-catalyzed reactive oxygen species generation, which leads to endothelial dysfunction and oxidative damage. Hemopexin (Hx) is a plasma heme scavenger able to prevent endothelial damage and tissue congestion in a model of heme overload. Here, we tested whether Hx could be used as a therapeutic tool to counteract heme toxic effects on the cardiovascular system in hemolytic diseases. Methods and Results— By using a model of heme overload in Hx-null mice, we demonstrated that heme excess in plasma, if not bound to Hx, promoted the production of reactive oxygen species and the induction of adhesion molecules and caused the reduction of nitric oxide availability. Then, we used &bgr;-thalassemia and sickle cell disease mice as models of hemolytic diseases to evaluate the efficacy of an Hx-based therapy in the treatment of vascular dysfunction related to heme overload. Our data demonstrated that Hx prevented heme-iron loading in the cardiovascular system, thus limiting the production of reactive oxygen species, the induction of adhesion molecules, and the oxidative inactivation of nitric oxide synthase/nitric oxide, and promoted heme recovery and detoxification by the liver mainly through the induction of heme oxygenase activity. Moreover, we showed that in sickle cell disease mice, endothelial activation and oxidation were associated with increased blood pressure and altered cardiac function, and the administration of exogenous Hx was found to almost completely normalize these parameters. Conclusions— Hemopexin treatment is a promising novel therapy to protect against heme-induced cardiovascular dysfunction in hemolytic disorders.

NADPH oxidases in heart failure: poachers or gamekeepers?

SIGNIFICANCE Oxidative stress is involved in the pathogenesis of heart failure but clinical antioxidant trials have been unsuccessful. This may be because effects of reactive oxygen species (ROS) depend upon their source, location, and concentration. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) proteins generate ROS in a highly regulated fashion and modulate several components of the heart failure phenotype. RECENT ADVANCES Two Nox isoforms, Nox2 and Nox4, are expressed in the heart. Studies using gene-modified mice deficient in Nox2 activity indicate that Nox2 activation contributes to angiotensin II-induced cardiomyocyte hypertrophy, atrial fibrillation, and the development of interstitial fibrosis but may also positively modulate physiological excitation-contraction coupling. Nox2 contributes to myocyte death under stress situations and plays important roles in postmyocardial infarction remodeling, in part by modulating matrix metalloprotease activity. In contrast to Nox2, Nox4 is constitutively active at a low level and induces protective effects in the heart under chronic stress, for example, by maintaining myocardial capillary density. However, high levels of Nox4 could have detrimental effects. CRITICAL ISSUES The effects of Nox proteins during the development of heart failure likely depend upon the isoform, activation level, and cellular distribution, and may include beneficial as well as detrimental effects. More needs to be learnt about the precise regulation of abundance and biochemical activity of these proteins in the heart as well as the downstream signaling pathways that they regulate. FUTURE DIRECTIONS The development of specific approaches to target individual Nox isoforms and/or specific cell types may be important for the achievement of therapeutic efficacy in heart failure.

Taming the PI3K team to hold inflammation and cancer at bay.

Recent progress in understanding the molecular mechanisms of receptor signal transduction is continuously highlighting new unforeseen potential drug targets for yet unmet therapeutic needs. While the large number of different cell surface receptors challenge the concept of antagonists development, the finding of signal transduction platforms common to multiple receptor families has boosted the development of new therapeutic approaches. The identification of the role of phosphoinositide 3-kinase family members downstream receptors as directors of multiple cellular responses ranging from cell proliferation and survival to immunity and cardiovascular control, is an example of successful drug target validation studies. This review will focus on these findings and on the ongoing efforts to tame this family of enzymes to beat inflammation and cancer.

Anchoring Proteins as Regulators of Signaling Pathways

Spatial and temporal organization of signal transduction is coordinated through the segregation of signaling enzymes in selected cellular compartments. This highly evolved regulatory mechanism ensures the activation of selected enzymes only in the vicinity of their target proteins. In this context, cAMP-responsive triggering of protein kinase A is modulated by a family of scaffold proteins referred to as A-kinase anchoring proteins. A-kinase anchoring proteins form the core of multiprotein complexes and enable simultaneous but segregated cAMP signaling events to occur in defined cellular compartments. In this review we will focus on the description of A-kinase anchoring protein function in the regulation of cardiac physiopathology.

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.

New signal transduction paradigms in anthracycline-induced cardiotoxicity.

Anthracyclines, such as doxorubicin, are the most potent and widely used chemotherapeutic agents for the treatment of a variety of human cancers, including solid tumors and hematological malignancies. However, their clinical use is hampered by severe cardiotoxic side effects and cancer therapy-related heart disease has become a leading cause of morbidity and mortality among cancer survivors. The identification of therapeutic strategies limiting anthracycline cardiotoxicity with preserved antitumor efficacy thus represents the current challenge of cardio-oncologists. Anthracycline cardiotoxicity has been originally ascribed to the ability of this class of drugs to disrupt iron metabolism and generate excess of reactive oxygen species (ROS). However, small clinical trials with iron chelators and anti-oxidants failed to provide any benefit and suggested that doxorubicin cardiotoxicity is not solely due to redox cycling. New emerging explanations include anthracycline-dependent regulation of major signaling pathways controlling DNA damage response, cardiomyocyte survival, cardiac inflammation, energetic stress and gene expression modulation. This review will summarize recent studies unraveling the complex web of mechanisms of doxorubicin-mediated cardiotoxicity, and identifying new druggable players for the prevention of heart disease in cancer patients. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.