Laura Pentassuglia

The role of Neuregulin-1β/ErbB signaling in the heart

Products of the Neuregulin-1 (Nrg-1) gene, along with the ErbB family of receptor tyrosine kinases through which Nrg-1 ligands signal, play a critical role during cardiovascular development. Through studies of genetically manipulated mice, as well as studies in cells isolated from adult hearts, it appears that Nrg-1/ErbB signaling is an essential paracrine mediator of cell-cell interactions that not only regulates tissue organization during development, but also helps to maintain cardiac function throughout an organisms life. Studies in cells isolated from the heart demonstrate that Nrg-1 can activate a number of signaling pathways, which mediate cellular adaptations to stress in the myocardium. These observations provide insight as to why ErbB2-targeted cancer treatments have deleterious effects on cardiac function in some cancer patients. Moreover emerging data suggest that Nrg-1 ligands might be useful clinically to restore cardiac function after cardiac injury. In this review we will attempt to synthesize the literature behind this rapidly growing and exciting area of research.

Inhibition of ErbB2 by receptor tyrosine kinase inhibitors causes myofibrillar structural damage without cell death in adult rat cardiomyocytes

Inhibition of ErbB2 (HER2) with monoclonal antibodies, an effective therapy in some forms of breast cancer, is associated with cardiotoxicity, the pathophysiology of which is poorly understood. Recent data suggest, that dual inhibition of ErbB1 (EGFR) and ErbB2 signaling is more efficient in cancer therapy, however, cardiac safety of this therapeutic approach is unknown. We therefore tested an ErbB1-(CGP059326) and an ErbB1/ErbB2-(PKI166) tyrosine kinase inhibitor in an in-vitro system of adult rat ventricular cardiomyocytes and assessed their effects on 1. cell viability, 2. myofibrillar structure, 3. contractile function, and 4. MAPK- and Akt-signaling alone or in combination with Doxorubicin. Neither CGP nor PKI induced cardiomyocyte necrosis or apoptosis. PKI but not CGP caused myofibrillar structural damage that was additive to that induced by Doxorubicin at clinically relevant doses. These changes were associated with an inhibition of excitation-contraction coupling. PKI but not CGP decreased p-Erk1/2, suggesting a role for this MAP-kinase signaling pathway in the maintenance of myofibrils. These data indicate that the ErbB2 signaling pathway is critical for the maintenance of myofibrillar structure and function. Clinical studies using ErbB2-targeted inhibitors for the treatment of cancer should be designed to include careful monitoring for cardiac dysfunction.

Disruption of a GATA4/Ankrd1 signaling axis in cardiomyocytes leads to sarcomere disarray: implications for anthracycline cardiomyopathy.

Doxorubicin (Adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin; however, the mechanism(s) of doxorubicin-induced CARP depletion and its specific role in cardiomyocytes have not been completely defined. We report that doxorubicin treatment in cardiomyocytes resulted in inhibition of CARP transcription, depletion of CARP protein levels, inhibition of myofilament gene transcription, and marked sarcomere disarray. Knockdown of CARP with small interfering RNA (siRNA) similarly inhibited myofilament gene transcription and disrupted cardiomyocyte sarcomere structure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. Doxorubicin also induced depletion of the cardiac transcription factor GATA4 in cardiomyocytes. CARP expression is regulated in part by GATA4, prompting us to examine the relationship between GATA4 and CARP in cardiomyocytes. We show in co-transfection experiments that GATA4 operates upstream of CARP by activating the proximal CARP promoter. GATA4-siRNA knockdown in cardiomyocytes inhibited CARP expression and myofilament gene transcription, and induced extensive sarcomere disarray. Adenoviral overexpression of GATA4 (AdV-GATA4) in cardiomyocytes prior to doxorubicin exposure maintained GATA4 levels, modestly restored CARP levels, and attenuated sarcomere disarray. Interestingly, siRNA-mediated depletion of CARP completely abolished the Adv-GATA4 rescue of the doxorubicin-induced sarcomere phenotype. These data demonstrate co-dependent roles for GATA4 and CARP in regulating sarcomere gene expression and maintaining sarcomeric organization in cardiomyocytes in culture. The data further suggests that concurrent depletion of GATA4 and CARP in cardiomyocytes by doxorubicin contributes in large part to myofibrillar disarray and the overall pathophysiology of anthracycline cardiomyopathy.

Intravenous Glial Growth Factor 2 (GGF2) Isoform of Neuregulin-1β Improves Left Ventricular Function, Gene and Protein Expression in Rats after Myocardial Infarction

Aims Recombinant Neuregulin (NRG)-1β has multiple beneficial effects on cardiac myocytes in culture, and has potential as a clinical therapy for heart failure (HF). A number of factors may influence the effect of NRG-1β on cardiac function via ErbB receptor coupling and expression. We examined the effect of the NRG-1β isoform, glial growth factor 2 (GGF2), in rats with myocardial infarction (MI) and determined the impact of high-fat diet as well as chronicity of disease on GGF2 induced improvement in left ventricular systolic function. Potential mechanisms for GGF2 effects on the remote myocardium were explored using microarray and proteomic analysis. Methods and Results Rats with MI were randomized to receive vehicle, 0.625 mg/kg, or 3.25 mg/kg GGF2 in the presence and absence of high-fat feeding beginning at day 7 post-MI and continuing for 4 weeks. Residual left ventricular (LV) function was improved in both of the GGF2 treatment groups compared with the vehicle treated MI group at 4 weeks of treatment as assessed by echocardiography. High-fat diet did not prevent the effects of high dose GGF2. In experiments where treatment was delayed until 8 weeks after MI, high but not low dose GGF2 treatment was associated with improved systolic function. mRNA and protein expression analysis of remote left ventricular tissue revealed a number of changes in myocardial gene and protein expression altered by MI that were normalized by GGF2 treatment, many of which are involved in energy production. Conclusions This study demonstrates that in rats with MI induced systolic dysfunction, GGF2 treatment improves cardiac function. There are differences in sensitivity of the myocardium to GGF2 effects when administered early vs. late post-MI that may be important to consider in the development of GGF2 in humans.

ErbB/integrin signaling interactions in regulation of myocardial cell-cell and cell-matrix interactions.

Neuregulin (Nrg)/ErbB and integrin signaling pathways are critical for the normal function of the embryonic and adult heart. Both systems activate several downstream signaling pathways, with different physiological outputs: cell survival, fibrosis, excitation-contraction coupling, myofilament structure, cell-cell and cell-matrix interaction. Activation of ErbB2 by Nrg1β in cardiomycytes or its overexpression in cancer cells induces phosphorylation of FAK (Focal Adhesion Kinase) at specific sites with modulation of survival, invasion and cell-cell contacts. FAK is also a critical mediator of integrin receptors, converting extracellular matrix alterations into intracellular signaling. Systemic FAK deletion is lethal and is associated with left ventricular non-compaction whereas cardiac restriction in adult hearts is well tolerated. Nevertheless, these hearts are more susceptible to stress conditions like trans-aortic constriction, hypertrophy, and ischemic injury. As FAK is both downstream and specifically activated by integrins and Nrg-1β, here we will explore the role of FAK in the heart as a protective factor and as possible mediator of the crosstalk between the ErbB and Integrin receptors. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Emerging Anticancer Therapeutic Targets and the Cardiovascular System: Is There Cause for Concern?

The race for a cure to cancer continues, fueled by unprecedented discoveries of fundamental biology underlying carcinogenesis and tumorigenesis. The expansion of the target list and tools to approach them is moving the oncology community extraordinarily rapidly to clinical trials, bringing new hope for cancer patients. This effort is also propelling biological discoveries in cardiovascular research, because many of the targets being explored in cancer play fundamental roles in the heart and vasculature. The combined efforts of cardiovascular and cancer biologists, along with clinical investigators in these fields, will be needed to understand how to safely exploit these efforts. Here, we discuss a few of the many research foci in oncology where we believe such collaboration will be particularly important.

Cardiac Side Effects of Anticancer Treatments: New Mechanistic Insights

Damage to heart cells leading to heart failure is a known complication of well-established cancer therapies including anthracycline antibiotics and radiation therapy, and the cardiovascular complications of these therapies has been controlled in large part through dose limitations and modifications of delivery methods. Recent research into the cellular and molecular mechanisms for the cardiovascular effects of these therapies may lead to other cardioprotective strategies that improve effectiveness of cancer treatments. Newer cancer therapies that have been developed based upon specifically targeting oncogene signaling also have been associated with heart failure. Rapid development of a detailed understanding of how these agents cause cardiac dysfunction promises to improve outcomes in cancer patients, as well as stimulate concepts of cardiovascular homeostasis that will likely accelerate development of cardiovascular therapies.

ErbB4 localization to cardiac myocyte nuclei, and its role in myocyte DNA damage response.

The intracellular domain of ErbB4 receptor tyrosine kinase is known to translocate to the nucleus of cells where it can regulate p53 transcriptional activity. The purpose of this study was to examine whether ErbB4 can localize to the nucleus of adult rat ventricular myocytes (ARVM), and regulate p53 in these cells. We demonstrate that ErbB4 does locate to the nucleus of cardiac myocytes as a full-length protein, although nuclear location occurs as a full-length protein that does not require Protein Kinase C or γ-secretase activity. Consistent with this we found that only the non-cleavable JM-b isoform of ErbB4 is expressed in ARVM. Doxorubicin was used to examine ErbB4 role in regulation of a DNA damage response in ARVM. Doxorubicin induced p53 and p21 was suppressed by treatment with AG1478, an EGFR and ErbB4 kinase inhibitor, or suppression of ErbB4 expression with small interfering RNA. Thus ErbB4 localizes to the nucleus as a full-length protein, and plays a role in the DNA damage response induced by doxorubicin in cardiac myocytes.

Neuregulin-1β regulation of embryonic endothelial progenitor cell survival

Endothelial progenitor cells (EPCs) are mobilized into the vascular space and home to damaged tissues, where they promote repair in part through a process of angiogenesis. Neuregulins (NRGs) are ligands in the epidermal growth factor family that signal through type I receptor tyrosine kinases in the erbB family (erbB2, erbB3, and erbB4) and regulate endothelial cell biology, promoting angiogenesis. Stimuli such as ischemia and exercise that promote EPC mobilization also induce cleavage and release of transmembrane NRG from cardiac microvascular endothelial cells (CMECs). We hypothesized that NRG/erbB signaling may regulate EPC biology. Using an embryonic (e)EPC cell line that homes to and repairs injured myocardium, we were able to detect erbB2 and erbB3 transcripts. Identical receptor expression was found in EPCs isolated from rat bone marrow and human whole blood. NRG treatment of eEPCs induces phosphorylation of kinases including Akt, GSK-3β, and Erk1/2 and the nuclear accumulation and transcriptional activation of β-catenin. NRG does not induce eEPC proliferation or migration but does protect eEPCs against serum deprivation-induced apoptosis. These results suggest a role for tissue-derived NRG in the regulation of EPC survival.

Cardiac mTOR complex 2 preserves ventricular function in pressure-overload hypertrophy

AIMS Mammalian target of rapamycin (mTOR), a central regulator of growth and metabolism, has tissue-specific functions depending on whether it is part of mTOR complex 1 (mTORC1) or mTORC2. We have previously shown that mTORC1 is required for adaptive cardiac hypertrophy and maintenance of function under basal and pressure-overload conditions. In the present study, we aimed to identify functions of mTORC2 in the heart. METHODS AND RESULTS Using tamoxifen-inducible cardiomyocyte-specific gene deletion, we generated mice deficient for cardiac rapamycin-insensitive companion of mTOR (rictor), an essential and specific component of mTORC2. Under basal conditions, rictor deficiency did not affect cardiac growth and function in young mice and also had no effects in adult mice. However, transverse aortic constriction caused dysfunction in the rictor-deficient hearts, whereas function was maintained in controls after 1 week of pressure overload. Adaptive increases in cardiac weight and cardiomyocyte cross-sectional area, fibrosis, and hypertrophic and metabolic gene expression were not different between the rictor-deficient and control mice. In control mice, maintained function was associated with increased protein levels of rictor, protein kinase C (PKC)βII, and PKCδ, whereas rictor ablation abolished these increases. Rictor deletion also significantly decreased PKCε at baseline and after pressure overload. Our data suggest that reduced PKCε and the inability to increase PKCβII and PKCδ abundance are, in accordance with their known function, responsible for decreased contractile performance of the rictor-deficient hearts. CONCLUSION Our study demonstrates that mTORC2 is implicated in maintaining contractile function of the pressure-overloaded male mouse heart.