James Cimino

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.

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.

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.

Hemopexin counteracts systolic dysfunction induced by heme-driven oxidative stress

Abstract Heart failure is a leading cause of morbidity and mortality in patients affected by different disorders associated to intravascular hemolysis. The leading factor is the presence of pathologic amount of pro‐oxidant free heme in the bloodstream, due to the exhaustion of the natural heme scavenger Hemopexin (Hx). Here, we evaluated whether free heme directly affects cardiac function, and tested the therapeutic potential of replenishing serum Hx for increasing serum heme buffering capacity. The effect of heme on cardiac function was assessed in vitro, on primary cardiomyocytes and H9c2 myoblast cell line, and in vivo, in Hx‐/‐ mice and in genetic and acquired mouse models of intravascular hemolysis. Purified Hx or anti‐oxidants N‐Acetyl‐L‐cysteine and &agr;‐tocopherol were used to counteract heme cardiotoxicity. In mice, Hx loss/depletion resulted in heme accumulation and enhanced reactive oxygen species (ROS) production in the heart, which ultimately led to severe systolic dysfunction. Similarly, high ROS reduced systolic Ca2+ transient amplitudes and fractional shortening in primary cardiomyocytes exposed to free heme. In keeping with these Ca2+ handling alterations, oxidation and CaMKII‐dependent phosphorylation of Ryanodine Receptor 2 were higher in Hx‐/‐ hearts than in controls. Administration of anti‐oxidants prevented systolic failure both in vitro and in vivo. Intriguingly, Hx rescued contraction defects of heme‐treated cardiomyocytes and preserved cardiac function in hemolytic mice. We show that heme‐mediated oxidative stress perturbs cardiac Ca2+ homeostasis and promotes contractile dysfunction. Scavenging heme, Hx counteracts cardiac heme toxicity and preserves left ventricular function. Our data generate the rationale to consider the therapeutic use of Hx to limit the cardiotoxicity of free heme in hemolytic disorders. Graphical abstract Figure. No Caption available. HighlightsIn hemolytic conditions, non‐hemopexin bound heme (NHBH) accumulates in the heart.In the heart, heme‐driven ROS affect Ca2+ homeostasis and impair systolic function.Hemopexin, by binding extracellular heme, prevents/limits heme cardiotoxicity.Hemolytic patients could have benefits from a therapy with heme chelators.

Phosphoinositide 3-Kinase Gamma Inhibition Protects from Anthracycline Cardiotoxicity and Reduces Tumor Growth.

Background: Anthracyclines, such as doxorubicin (DOX), are potent anticancer agents for the treatment of solid tumors and hematologic malignancies. However, their clinical use is hampered by cardiotoxicity. This study sought to investigate the role of phosphoinositide 3-kinase &ggr; (PI3K&ggr;) in DOX-induced cardiotoxicity and the potential cardioprotective and anticancer effects of PI3K&ggr; inhibition. Methods: Mice expressing a kinase-inactive PI3K&ggr; or receiving PI3K&ggr;-selective inhibitors were subjected to chronic DOX treatment. Cardiac function was analyzed by echocardiography, and DOX-mediated signaling was assessed in whole hearts or isolated cardiomyocytes. The dual cardioprotective and antitumor action of PI3K&ggr; inhibition was assessed in mouse mammary tumor models. Results: PI3K&ggr; kinase-dead mice showed preserved cardiac function after chronic low-dose DOX treatment and were protected against DOX-induced cardiotoxicity. The beneficial effects of PI3K&ggr; inhibition were causally linked to enhanced autophagic disposal of DOX-damaged mitochondria. Consistently, either pharmacological or genetic blockade of autophagy in vivo abrogated the resistance of PI3K&ggr; kinase-dead mice to DOX cardiotoxicity. Mechanistically, PI3K&ggr; was triggered in DOX-treated hearts, downstream of Toll-like receptor 9, by the mitochondrial DNA released by injured organelles and contained in autolysosomes. This autolysosomal PI3K&ggr;/Akt/mTOR/Ulk1 signaling provided maladaptive feedback inhibition of autophagy. PI3K&ggr; blockade in models of mammary gland tumors prevented DOX-induced cardiac dysfunction and concomitantly synergized with the antitumor action of DOX by unleashing anticancer immunity. Conclusions: Blockade of PI3K&ggr; may provide a dual therapeutic advantage in cancer therapy by simultaneously preventing anthracyclines cardiotoxicity and reducing tumor growth.

Cardiac concentric hypertrophy promoted by activated Met receptor is mitigated in vivo by inhibition of Erk1,2 signalling with Pimasertib

Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. In this work, we unveiled the events associated with cardiac hypertrophy by means of a transgenic model expressing activated Met receptor. c-Met proto-oncogene encodes for the tyrosine kinase receptor of Hepatocyte growth factor and is a strong inducer of Ras-Raf-Mek-Erk1,2 pathway. We showed that three weeks after the induction of activated Met, the heart presents a remarkable concentric hypertrophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement is accompanied by upregulation of growth-regulating transcription factors, natriuretic peptides, cytoskeletal proteins, and Extracellular Matrix remodelling factors (Timp1 and Pai1). At a later stage, cardiac hypertrophic remodelling results into heart failure with preserved systolic function. Prevention trial by suppressing activated Met showed that cardiac hypertrophy is reversible, and progression to heart failure is prevented. Notably, treatment with Pimasertib, Mek1 inhibitor, attenuates cardiac hypertrophy and remodelling. Our results suggest that modulation of Erk1.2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies.

A New Transgenic Mouse Model of Heart Failure and Cardiac Cachexia Raised by Sustained Activation of Met Tyrosine Kinase in the Heart

Among other diseases characterized by the onset of cachexia, congestive heart failure takes a place of relevance, considering the high prevalence of this pathology in most European countries and in the United States, and is undergoing a rapid increase in developing countries. Actually, only few models of cardiac cachexia exist. Difficulties in the recruitment and follow-up of clinical trials implicate that new reproducible and well-characterized animal models are pivotal in developing therapeutic strategies for cachexia. We generated a new model of cardiac cachexia: a transgenic mouse expressing Tpr-Met receptor, the activated form of c-Met receptor of hepatocyte growth factor, specifically in the heart. We showed that the cardiac-specific induction of Tpr-Met raises a cardiac hypertrophic remodelling, which progresses into concentric hypertrophy with concomitant increase in Gdf15 mRNA levels. Hypertrophy progresses to congestive heart failure with preserved ejection fraction, characterized by reduced body weight gain and food intake and skeletal muscle wasting. Prevention trial by suppressing Tpr-Met showed that loss of body weight could be prevented. Skeletal muscle wasting was also associated with altered gene expression profiling. We propose transgenic Tpr-Met mice as a new model of cardiac cachexia, which will constitute a powerful tool to understand such complex pathology and test new drugs/approaches at the preclinical level.

Data demonstrating the anti-oxidant role of hemopexin in the heart

The data presented in this article are related to the research article entitled Hemopexin counteracts systolic dysfunction induced by heme-driven oxidative stress (G. Ingoglia, C. M. Sag, N. Rex, L. De Franceschi, F. Vinchi, J. Cimino, S. Petrillo, S. Wagner, K. Kreitmeier, L. Silengo, F. Altruda, L. S. Maier, E. Hirsch, A. Ghigo and E. Tolosano, 2017) [1]. Data show that heme induces reactive oxygen species (ROS) production in primary cardiomyocytes. H9c2 myoblastic cells treated with heme bound to human Hemopexin (Hx) are protected from heme accumulation and oxidative stress. Similarly, the heme-driven oxidative response is reduced in primary cardiomyocytes treated with Hx-heme compared to heme alone. Our in vivo data show that mouse models of hemolytic disorders, β-thalassemic mice and phenylhydrazine-treated mice, have low serum Hx associated to enhanced expression of heme- and oxidative stress responsive genes in the heart. Hx-/- mice do not show signs of heart fibrosis or overt inflammation. For interpretation and discussion of these data, refer to the research article referenced above.