Monica Palmeri

AMP-activated protein kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury

AMP-activated protein kinase (AMPK) is an important regulator of diverse cellular pathways in the setting of energetic stress. Whether AMPK plays a critical role in the metabolic and functional responses to myocardial ischemia and reperfusion remains uncertain. We examined the cardiac consequences of long-term inhibition of AMPK activity in transgenic mice expressing a kinase dead (KD) form of the enzyme. The KD mice had normal fractional shortening and no heart failure, cardiac hypertrophy, or fibrosis, although the in vivo left ventricular (LV) dP/dt was lower than that in WT hearts. During low-flow ischemia and postischemic reperfusion in vitro, KD hearts failed to augment glucose uptake and glycolysis, although glucose transporter content and insulin-stimulated glucose uptake were normal. KD hearts also failed to increase fatty acid oxidation during reperfusion. Furthermore, KD hearts demonstrated significantly impaired recovery of LV contractile function during postischemic reperfusion that was associated with a lower ATP content and increased injury compared with WT hearts. Caspase-3 activity and TUNEL-staining were increased in KD hearts after ischemia and reperfusion. Thus, AMPK is responsible for activation of glucose uptake and glycolysis during low-flow ischemia and plays an important protective role in limiting damage and apoptotic activity associated with ischemia and reperfusion in the heart.

Endothelium-Derived Neuregulin Protects the Heart Against Ischemic Injury

Background— Removal of cardiac endothelial cells (EC) has been shown to produce significant detrimental effects on the function of adjacent cardiac myocytes, suggesting that EC play a critical role in autocrine/paracrine regulation of the heart. Despite this important observation, the mediators of the protective function of EC remain obscure. Neuregulin (NRG, a member of the epidermal growth factor family) is produced by EC and cardiac myocytes contain receptors (erbB) for this ligand. We hypothesized that NRG is an essential factor produced by EC, which promotes cardioprotection against ischemic injury. Methods and Results— We demonstrate that human cardiac EC express and release NRG in response to hypoxia–reoxygenation. Under conditions where hypoxia–reoxygenation causes significant cardiac myocyte cell death, NRG can significantly decrease apoptosis of isolated adult ventricular myocytes. Coculturing adult murine myocytes with human umbilical vein, murine lung microvascular, or human coronary artery EC can also protect myocytes against hypoxia–reoxygenation–induced apoptosis. These protective effects are abolished by NRG gene deletion or silencing of NRG expression in EC. Finally, endothelium-selective deletion of NRG in vivo leads to significantly decreased tolerance to ischemic insult, as demonstrated by impaired postischemic contractile recovery in a perfused whole-organ preparation and larger infarct sizes after coronary artery ligation. Conclusion— Together, these data demonstrate that EC-derived NRG plays an important role in cardiac myocyte protection against ischemic injury in the heart and supports the idea that manipulation of this signaling pathway may be an important clinical target in this setting.

Role of uncoupling protein 3 in ischemia-reperfusion injury, arrhythmias, and preconditioning

Overexpression of mitochondrial uncoupling proteins (UCPs) attenuates ischemia-reperfusion (I/R) injury in cultured cardiomyocytes. However, it is not known whether UCPs play an essential role in cardioprotection in the intact heart. This study evaluated the cardioprotective efficacy of UCPs against I/R injury and characterized the mechanism of UCP-mediated protection in addition to the role of UCPs in ischemic preconditioning (IPC). Cardiac UCP3 knockout (UCP3(-/-)) and wild-type (WT) mice hearts were subjected to ex vivo and in vivo models of I/R injury and IPC. Isolated UCP3(-/-) mouse hearts were retrogradely perfused and found to have poorer recovery of left ventricular function compared with WT hearts under I/R conditions. In vivo occlusion of the left coronary artery resulted in twofold larger infarcts in UCP3(-/-) mice compared with WT mice. Moreover, the incidence of in vivo I/R arrhythmias was higher in UCP3(-/-) mice. Myocardial energetics were significantly impaired with I/R, as reflected by a decreased ATP content and an increase in the AMP-to-ATP ratio. UCP3(-/-) hearts generated more reactive oxygen species (ROS) than WT hearts during I/R. Pretreatment of UCP3(-/-) hearts with the pharmacological uncoupling agent carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone improved postischemic functional recovery. Also the protective efficacy of IPC was abolished in UCP3(-/-) mice. We conclude that UCP3 plays a critical role in cardioprotection against I/R injury and the IPC phenomenon. There is increased myocardial vulnerability to I/R injury in hearts lacking UCP3. The mechanisms of UCP3-mediated cardioprotection include regulation of myocardial energetics and ROS generation by UCP3 during I/R.

AMPK is critical for mitochondrial function during reperfusion after myocardial ischemia.

AMP-activated kinase (AMPK) is a stress responsive kinase that regulates cellular metabolism and protects against cardiomyocyte injury during ischemia-reperfusion (IR). Mitochondria play an important role in cell survival, but the specific actions of activated AMPK in maintaining mitochondrial integrity and function during reperfusion are unknown. Thus, we assessed the consequences of AMPK inactivation on heart mitochondrial function during reperfusion. Mouse hearts expressing wild type (WT) or kinase-dead (KD) AMPK were studied. Mitochondria isolated from KD hearts during reperfusion had intact membrane integrity, but demonstrated reduced oxidative capacity, increased hydrogen peroxide production and decreased resistance to mitochondrial permeability transition pore opening compared to WT. KD hearts showed increased activation of the mitogen activated protein kinase kinase 4 (MKK4) and downstream c-Jun terminal kinase (JNK) and greater necrosis during reperfusion after coronary occlusion. Transgenic expression of mitochondrial catalase (MCAT) prevented the excessive cardiac JNK activation and attenuated the increased myocardial necrosis observed during reperfusion in KD mice. Inhibition of JNK increased the resistance of KD hearts to mPTP opening, contractile dysfunction and necrosis during IR. Thus, intrinsic activation of AMPK is critical to prevent excess mitochondrial reactive oxygen production and consequent JNK signaling during reperfusion, thereby protecting against mPTP opening, irreversible mitochondrial damage and myocardial injury.

Activation of the aryl hydrocarbon receptor by doxorubicin mediates cytoprotective effects in the heart

AIMS Doxorubicin (DOX) is a highly effective chemotherapeutic agent; however, cumulative dose-dependent cardiotoxicity is a significant side effect of this therapy. Because DOX is a polyaromatic hydrocarbon, we hypothesized that it will be metabolized by the activation of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that is involved in the metabolism of numerous xenobiotic agents. These studies were performed to determine whether DOX activates AhR and whether this activation modulates the toxicity of DOX in cardiomyocytes. METHODS AND RESULTS Treatment with DOX induced AhR migration to the nucleus, increased AhR binding with its co-factor, aryl hydrocarbon receptor nuclear translocator-1 (ARNT1), and increased the expression of AhR-regulated phase I (CYP1A1) and phase II (GSTA1) drug-metabolizing enzymes in both cardiomyocytes and in the intact heart. Knockdown of AhR in H9C2 cells abolished DOX-induced increases in CYP1A1 and GSTA1 expression. Similar results were obtained by treating adult rat ventricular myocytes with the AhR antagonist, CH-223191. Taken together, these findings indicate that DOX-induced upregulation of CYP1A1 and GSTA1 expression is AhR dependent. AhR null mice treated with 10 mg/kg DOX did not show any activation of CYP1A1 or GSTA1 expression. Moreover, lack of AhR in vivo resulted in a significant decrease in left ventricular function compared with wild-type animals, and increased p53 activation and apoptosis in the heart after treatment with DOX. CONCLUSIONS These findings indicate that AhR plays an important role in DOX metabolism by the heart and further demonstrate that AhR is cardioprotective against DOX-induced cardiotoxicity.

Abstract C227: Doxorubicin induces AKT phosphorylation through activation of the aryl hydrocarbon receptor in the heart

Doxorubicin (DOX) is one of the most effective anticancer drugs in treating different types of tumors. However, cumulative cardiotoxicity is a significant side effect of this therapy resulting in clinical heart failure through the production of reactive oxygen species (ROS). However, the protective responses of the heart to DOX treatment are still unknown. Aryl hydrocarbon receptor (AhR) is a ligand‐activated transcription factor that is primarily involved in the detoxification of xenobiotic agents that generate ROS. Activation of AhR results in its migration to the nucleus where it dimerizes with ARNT1 to form a transcription factor for genes coding many phase I drug metabolizing enzymes, such as the cytochrome P450, CYP1A1. We hypothesize that DOX treatment activates AhR and triggers Akt phosphorylation as a protective mechanism against oxidant stress. H9C2 cells incubated with 2.5 M DOX for 4 hours showed a significant increase of AhR protein levels in the nuclear fraction, which then decreased to baseline levels by 24 hours of treatment. The same pattern of change was seen for CYP1A1 expression. Immunoprecipitation of the nuclear protein fraction with AhR antibodies and subsequent immunoblotting with ARNT1 antibodies showed increased binding between these proteins after 4 hours of DOX treatment. Similar results were shown when ARNT1 immunoprecipitation and AhR immunoblotting were performed. Increased phosphorylation of Akt at Ser473 was detected at 4 hours of DOX treatment, which decreased by 24 hours. A similar temporal pattern of phosphorylation was identified for known downstream targets of Akt (pSer9‐GSK3 and pSer136‐BAD). Transfection with either AhR or ARNT1 siRNAs blocked the phosphorylation of Akt following DOX treatment in H9C2 cells, increased ROS production as well as cytotoxicity. DOX treatment of isolated adult rat cardiomyocytes showed similar pattern of changes in Akt phosphorylation and its downstream targets as well as increased oxidative stress. In mice injected with 10 mg/kg DOX, AhR translocated into the nucleus by 4 hours after injection, which was associated with ARNT1 binding and increased CYP1A1 expression. Phosphorylation of Akt, GSK3 and BAD increased by 24 hours after DOX injection. Oxidative stress was significantly higher in mice injected with DOX after 4 hours. We conclude that the early cardiac response to DOX treatment involves Akt phosphorylation through AhR activation.Moreover, silencing of AhR might lead to higher oxidative stress and increased cellular toxicity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C227.