Stephanie Pesant

Cardiotoxicity of the cancer therapeutic agent imatinib mesylate

Imatinib mesylate (Gleevec) is a small-molecule inhibitor of the fusion protein Bcr-Abl, the causal agent in chronic myelogenous leukemia. Here we report ten individuals who developed severe congestive heart failure while on imatinib and we show that imatinib-treated mice develop left ventricular contractile dysfunction. Transmission electron micrographs from humans and mice treated with imatinib show mitochondrial abnormalities and accumulation of membrane whorls in both vacuoles and the sarco- (endo-) plasmic reticulum, findings suggestive of a toxic myopathy. With imatinib treatment, cardiomyocytes in culture show activation of the endoplasmic reticulum (ER) stress response, collapse of the mitochondrial membrane potential, release of cytochrome c into the cytosol, reduction in cellular ATP content and cell death. Retroviral gene transfer of an imatinib-resistant mutant of c-Abl, alleviation of ER stress or inhibition of Jun amino-terminal kinases, which are activated as a consequence of ER stress, largely rescues cardiomyocytes from imatinib-induced death. Thus, cardiotoxicity is an unanticipated side effect of inhibition of c-Abl by imatinib.

Level of G protein–Coupled Receptor Kinase-2 Determines Myocardial Ischemia/Reperfusion Injury via Pro- and Anti-Apoptotic Mechanisms

Rationale: Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein–coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function. Objective: A loss of cardiac GRK2 activity is known to arrest progression of heart failure (HF), at least in part by normalization of cardiac &bgr;-adrenergic receptor (&bgr;AR) signaling. Chronic HF studies have been performed with GRK2 knockout mice, as well as expression of the &bgr;ARKct, a peptide inhibitor of GRK2 activity. This study was conducted to examine the role of GRK2 and its activity during acute myocardial ischemic injury using an I/R model. Methods and Results: We demonstrate, using cardiac-specific GRK2 and &bgr;ARKct-expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with &bgr;ARKct imparting cardioprotection. Post-I/R infarct size was greater in GRK2-overexpressing mice (45.0±2.8% versus 31.3±2.3% in controls) and significantly smaller in &bgr;ARKct mice (16.8±1.3%, P<0.05). Importantly, in vivo apoptosis was found to be consistent with these reciprocal effects on post-I/R myocardial injury when levels of GRK2 activity were altered. Moreover, these results were reflected by higher Akt activation and induction of NO production via &bgr;ARKct, and these antiapoptotic/survival effects could be recapitulated in vitro. Interestingly, selective antagonism of &bgr;2ARs abolished &bgr;ARKct-mediated cardioprotection, suggesting that enhanced GRK2 activity on this GPCR is deleterious to cardiac myocyte survival. Conclusion: The novel effect of reducing acute ischemic myocardial injury via increased Akt activity and NO production adds significantly to the therapeutic potential of GRK2 inhibition with the &bgr;ARKct not only in chronic HF but also potentially in acute ischemic injury conditions.

Post-ischemic cardioprotection by A2A adenosine receptors: dependent of phosphatidylinositol 3-kinase pathway.

Activation of myocardial A2A adenosine receptors during reperfusion has been shown to be cardioprotective. The intracellular mechanisms underlying this protection remain unknown. To understand the beneficial effects of activated A2A adenosine receptors in such a state, we investigated whether the enzymes phosphatidylinositol 3-kinase (PI3K) and caspase-3 can account for this post-ischemic cardioprotective effect in an anesthetized rabbit model of myocardial infarction (30 minutes ischemia; 5 hours reperfusion). Administration of the A2A agonist CGS21680 (0.2 &mgr;g/kg/min) 5 minutes before reperfusion began (Early) reduced infarct size expressed as a percentage of the area at risk (25.7 ± 5.3% versus 46.5 ± 5.3% for the control group; * P < 0.05). Treatment with the A2A agonist 5 minutes after the onset of reperfusion (Late) had no effect on infarct size (38.2 ± 6.2%). In the presence of a selective inhibitor of PI3K (LY294002), the beneficial effects of CGS21680 on infarct size was no longer observed (43.9 ± 7.9%). After 5 hours of reperfusion, higher PI3K activity in the ischemic region was observed in the Early group compared with the other experimental groups. Caspase-3 activity was not observed in these different groups. In another set of experiments, PI3K activity was significantly higher during the first 15 minutes of reperfusion in the Early group as compared with the Control group. Caspase-3 activity increased rapidly during the first 15 minutes of reperfusion in the Control group and remained stable in the Early group. These results indicated that post-ischemic cardioprotection afforded by A2A adenosine receptor activation is PI3K-dependent and modulate rapidly other signaling pathways such as caspase-3.

GPCR signalling in hypertension: role of GRKs.

Hypertension is a prevalent condition in the developed world and disease severity is directly correlated with additional cardiovascular complications. It is estimated that 30% of the adult population in the United States has hypertension, which is classified as a systolic blood pressure > or =140 mmHg and/or a diastolic blood pressure > or =90 mmHg. A prolonged increase in afterload ultimately leads to congestive heart failure in the majority of cases. Currently, medication designed to treat hypertension is inadequate, thus new therapies need to be explored. Blood pressure is tightly regulated by blood vessel radius, which is established by hormones and/or peptides binding to GPCRs (G-protein-coupled receptors). Catecholamines and peptide hormones, such as AngII (angiotensin II), are elevated in hypertension and, therefore, signalling by these GPCRs is increased. Their signalling is tightly controlled by a class of proteins, the GRKs (GPCR kinases). Elevated levels of either GRK2 or GRK5 in both the lymphocytes and VSM (vascular smooth muscle) are associated with human hypertension and animal models of the disease. The focus of the present review is on the role GRKs, and their regulation of GPCRs, play in high blood pressure.

Inhibition of vascular smooth muscle G protein-coupled receptor kinase 2 enhances α1D-adrenergic receptor constriction

G protein-coupled receptor kinase 2 (GRK2) is a serine/theorinine kinase that phosphorylates and desensitizes agonist-bound G protein-coupled receptors. GRK2 is increased in expression and activity in lymphocytes and vascular smooth muscle (VSM) in human hypertension and animal models of the disease. Inhibition of GRK2 using the carboxyl-terminal portion of the protein (GRK2ct) has been an effective tool to restore compromised beta-adrenergic receptor (AR) function in heart failure and improve outcome. A well-characterized dysfunction in hypertension is attenuation of betaAR-mediated vasodilation. Therefore, we tested the role of inhibition of GRK2 using GRK2ct or VSM-selective GRK2 gene ablation in a renal artery stenosis model of elevated blood pressure (BP) [the two-kidney, one-clip (2K1C) model]. Use of the 2K1C model resulted in a 30% increase in conscious BP, a threefold increase in plasma norepinephrine levels, and a 50% increase in VSM GRK2 mRNA levels. BP remained increased despite VSM-specific GRK2 inhibition by either GRK2 knockout (GRK2KO) or peptide inhibition (GRK2ct). Although betaAR-mediated dilation in vivo and in situ was enhanced, alpha(1)AR-mediated vasoconstriction was also increased. Further pharmacological experiments using alpha(1)AR antagonists revealed that GRK2 inhibition of expression (GRK2KO) or activity (GRK2ct) enhanced alpha(1D)AR vasoconstriction. This is the first study to suggest that VSM alpha(1D)ARs are a GRK2 substrate in vivo.

Simultaneous Administration of Insulin-Like Growth Factor-1 and Darbepoetin Alfa Protects the Rat Myocardium Against Myocardial Infarction and Enhances Angiogenesis

Recent studies have shown that insulin growth factor‐1 (IGF‐1) and either erythropoietin (EPO) or the long‐acting EPO analog Darbepoetin alfa (DA) protect the heart against ischemia/reperfusion (I/R) and myocardial infarction (MI). The present study examined the cardioprotective effect of simultaneous treatments with IGF‐1 and DA in these models of cardiac injury. Rats were subjected to I/R or MI and were treated with IGF‐1, DA, and a combination of IGF‐1 and DA, or vehicle treatment. IGF‐1 and DA treatments imparted similar protective effect by reducing infarct size. Moreover, these treatments led to improvement of cardiac function after I/R or MI compared to vehicle. In the reperfused heart, apoptosis was reduced with either or both IGF‐1 and DA treatments as measured by reduced TUNEL staining and caspase‐3 activity. In addition, after MI, treatment with IGF‐1 or DA significantly induced angiogenesis. This angiogenic effect was enhanced significantly when IGF‐1 and DA were given simultaneously compared to vehicle or either agents alone. These data indicate simultaneous pharmacological treatments with IGF‐1 and DA protect the heart against I/R and MI injuries. This protection results in reduced infarct size and improved cardiac function. Moreover, this treatment reduces apoptosis and enhances angiogenesis in the ischemic heart.

Inhibition of Angiotensin II Gq Signaling Augments β-Adrenergic Receptor Mediated Effects in a Renal Artery Stenosis Model of High Blood Pressure

Chronic ventricular pressure overload states, such as hypertension, and elevated levels of neurohormones (norepinephrine, angiotensin II, endothelin-1) initiate cardiac hypertrophy and dysfunction and share the property of being able to bind to Gq-coupled 7-transmembrane receptors. The goal of the current study was to determine the role of endogenous cardiac myocyte Gq signaling and its role in cardiac hypertrophy and dysfunction during high blood pressure (BP). We induced renal artery stenosis for 8 weeks in control mice and mice expressing a peptide inhibitor of Gq signaling (GqI) using a 2 kidney, 1 clip renal artery stenosis model. 8 weeks following chronic high BP, control mice had cardiac hypertrophy and depressed function. Inhibition of cardiomyocyte Gq signaling did not reverse cardiac hypertrophy but attenuated increases in a profile of cardiac profibrotic genes and genes associated with remodeling. Inhibition of Gq signaling also attenuated the loss of cardiac function. We determined that Gq signaling downstream of angiotensin II receptor stimulation negatively impacted beta-adrenergic receptor (AR) responses and inhibition of Gq signaling was sufficient to restore betaAR-mediated responses. Therefore, in this study we found that Gq signaling negatively impacts cardiac function during high BP. Specifically, we found that inhibition of AT1-Gq signaling augmented betaAR mediated effects in a renal artery stenosis model of hypertension. These observations may underlie additional, beneficial effects of angiotensinogen converting enzyme (ACE) inhibitors and angiotensin receptor antagonists observed during times of hemodynamic stress.

Negative Regulation of VEGF Signaling in Human Coronary Artery Endothelial Cells by G Protein‐Coupled Receptor Kinase 5

G protein‐coupled receptor kinase 5 (GRK5) is present in endothelial cells (ECs) and has the potential to regulate EC function through seven transmembrane‐spanning receptor (7TMR) signaling. Recently, it has been appreciated that GRKs can affect receptor tyrosine kinases (RTKs). VEGF, an RTK, is one of the most potent mediators for EC function and angiogenesis; therefore, we determined the role GRK5 plays in VEGF signaling in human coronary artery ECs (HCAECs). GRK5 levels were increased by VEGF treatment in HCAECs. Adenoviral overexpression of GRK5 inhibited migration and proliferation of HCAECs in response to VEGF. GRK5 overexpression in HCAECs significantly suppressed both acute and late activation of Akt and extracellular signal‐related kinase (ERKs) as well as the phosphorylationof GSK‐3β, an endogenous substrate of Akt. Coimmunoprecipitations revealed that GRK5 is physically associated with Akt. This study shows for the first time that GRK5 negatively regulates VEGF signaling in HCAECs and suggests that targeted intervention of GRK5 in ECs might be a novel therapeutic strategy to prevent and treat disorders involving altered EC function.