Xiaoyue Hu

Low-Flow Ischemia Leads to Translocation of Canine Heart GLUT-4 and GLUT-1 Glucose Transporters to the Sarcolemma In Vivo

BACKGROUND Myocardial ischemia increases heart glucose utilization in vivo. However, whether low-flow ischemia leads to the translocation of glucose transporter (GLUT)-4 and/or GLUT-1 to the sarcolemma in vivo is unknown. METHODS AND RESULTS In a canine model, we evaluated myocardial glucose metabolism in vivo and the distribution of GLUT-4 and GLUT-1 by use of immunoblotting of sarcolemma and intracellular membranes and immunofluorescence localization with confocal microscopy. In vivo glucose extraction increased fivefold (P < .001) and was associated with net lactate release in the ischemic region. Ischemia led to an increase in the sarcolemma content of both GLUT-4 (15 +/- 2% to 30 +/- 3%, P < .02) and GLUT-1 (41 +/- 4% to 58 +/- 3%, P < .03) compared with the nonischemic region and to a parallel decrease in their intracellular contents. Immunofluorescence demonstrated the presence of both GLUT-4 and GLUT-1 on cardiac myocytes. GLUT-1 had a more prominent cell surface pattern than GLUT-4, which was primarily intracellular in the nonischemic region. However, significant GLUT-4 surface labeling was found in the ischemic region. CONCLUSIONS Translocation of the insulin-responsive GLUT-4 transporter from an intracellular storage pool to the sarcolemma occurs in vivo during acute low-flow ischemia. GLUT-1 is also present in an intracellular storage pool from which it undergoes translocation to the sarcolemma in response to ischemia. These results indicate that both GLUT-1 and GLUT-4 are important in ischemia-mediated myocardial glucose uptake in vivo.

Cardiac macrophage migration inhibitory factor inhibits JNK pathway activation and injury during ischemia/reperfusion

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that also modulates physiologic cell signaling pathways. MIF is expressed in cardiomyocytes and limits cardiac injury by enhancing AMPK activity during ischemia. Reperfusion injury is mediated in part by activation of the stress kinase JNK, but whether MIF modulates JNK in this setting is unknown. We examined the role of MIF in regulating JNK activation and cardiac injury during experimental ischemia/reperfusion in mouse hearts. Isolated perfused Mif-/- hearts had greater contractile dysfunction, necrosis, and JNK activation than WT hearts, with increased upstream MAPK kinase 4 phosphorylation, following ischemia/reperfusion. These effects were reversed if recombinant MIF was present during reperfusion, indicating that MIF deficiency during reperfusion exacerbated injury. Activated JNK acts in a proapoptotic manner by regulating BCL2-associated agonist of cell death (BAD) phosphorylation, and this effect was accentuated in Mif-/- hearts after ischemia/reperfusion. Similar detrimental effects of MIF deficiency were observed in vivo following coronary occlusion and reperfusion in Mif-/- mice. Importantly, excess JNK activation also was observed after hypoxia-reoxygenation in human fibroblasts homozygous for the MIF allele with the lowest level of promoter activity. These data indicate that endogenous MIF inhibits JNK pathway activation during reperfusion and protects the heart from injury. These findings have clinical implications for patients with the low-expression MIF allele.

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.

Additive Effects of Hyperinsulinemia and Ischemia on Myocardial GLUT1 and GLUT4 Translocation In Vivo

BACKGROUND Myocardial ischemia increases glucose uptake through the translocation of GLUT1 and GLUT4 from an intracellular compartment to the sarcolemma. The present study was performed to determine whether hyperinsulinemia causes translocation of myocardial GLUT1 as well as GLUT4 in vivo and whether there are additive effects of insulin and ischemia on GLUT1 and GLUT4 translocation. METHODS ADN RESULTS: Myocardial glucose uptake and transporter distribution were assessed by arteriovenous measurements, cell fractionation, and immunofluorescence. In fasted anesthetized dogs, hyperinsulinemia increased myocardial glucose extraction 3-fold (P<0.01) and the sarcolemmal content of GLUT4 by 90% and GLUT1 by 50% (P<0.05 for both) compared with saline infusion. In subsequent experiments, glucose uptake and transporter distribution were determined in ischemic and nonischemic regions of hearts from hyperinsulinemic animals during regional myocardial ischemia. Glucose uptake was 50% greater in the ischemic region (P<0.05). This was associated with a 20% increase in sarcolemmal GLUT1 and a 60% increase in sarcolemmal GLUT4 contents in the ischemic region (P<0.05 for both). CONCLUSIONS Insulin stimulates myocardial glucose utilization through translocation of GLUT1 as well as GLUT4. Insulin and ischemia have additive effects to increase in vivo glucose utilization and augment glucose transporter translocation. We conclude that recruitment of both GLUT1 and GLUT4 contributes to increased myocardial glucose uptake during moderate reductions in coronary blood flow under insulin-stimulated conditions.

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.

The vestigial enzyme D-dopachrome tautomerase protects the heart against ischemic injury

The cellular response to stress involves the recruitment and coordination of molecular signaling pathways that prevent cell death. D-dopachrome tautomerase (DDT) is an enzyme that lacks physiologic substrates in mammalian cells, but shares partial sequence and structural homology with macrophage migration inhibitory factor (MIF). Here, we observed that DDT is highly expressed in murine cardiomyocytes and secreted by the heart after ischemic stress. Antibody-dependent neutralization of secreted DDT exacerbated both ischemia-induced cardiac contractile dysfunction and necrosis. We generated cardiomyocyte-specific DDT knockout mice (Myh6-Cre Ddtfl/fl), which demonstrated normal baseline cardiac size and function, but had an impaired physiologic response to ischemia-reperfusion. Hearts from Myh6-Cre Ddtfl/fl mice exhibited more necrosis and LV contractile dysfunction than control hearts after coronary artery ligation and reperfusion. Furthermore, treatment with DDT protected isolated hearts against injury and contractile dysfunction after ischemia-reperfusion. The protective effect of DDT required activation of the metabolic stress enzyme AMP-activated protein kinase (AMPK), which was mediated by a CD74/CaMKK2-dependent mechanism. Together, our data indicate that cardiomyocyte secretion of DDT has important autocrine/paracrine effects during ischemia-reperfusion that protect the heart against injury.

Urocortin 2 autocrine/paracrine and pharmacologic effects to activate AMP-activated protein kinase in the heart

Urocortin 2 (Ucn2), a peptide of the corticotropin-releasing factor (CRF) family, binds with high affinity to type 2 CRF receptors (CRFR2) on cardiomyocytes and confers protection against ischemia/reperfusion. The mechanisms by which the Ucn2-CRFR2 axis mitigates against ischemia/reperfusion injury remain incompletely delineated. Activation of AMP-activated protein kinase (AMPK) also limits cardiac damage during ischemia/reperfusion. AMPK is classically activated by alterations in cellular energetics; however, hormones, cytokines, and additional autocrine/paracrine factors also modulate its activity. We examined the effects of both the endogenous cardiac Ucn2 autocrine/paracrine pathway and Ucn2 treatment on AMPK regulation. Ucn2 treatment increased AMPK activation and downstream acetyl-CoA carboxylase phosphorylation and glucose uptake in isolated heart muscles. These actions were blocked by the CRFR2 antagonist anti–sauvagine-30 and by a PKCε translocation-inhibitor peptide (εV1-2). Hypoxia-induced AMPK activation was also blunted in heart muscles by preincubation with either anti–sauvagine-30, a neutralizing anti-Ucn2 antibody, or εV1-2. Treatment with Ucn2 in vivo augmented ischemic AMPK activation and reduced myocardial injury and cardiac contractile dysfunction after regional ischemia/reperfusion in mice. Ucn2 also directly activated AMPK in ex vivo-perfused mouse hearts and diminished injury and contractile dysfunction during ischemia/reperfusion. Thus, both Ucn2 treatment and the endogenous cardiac Ucn2 autocrine/paracrine pathway activate AMPK signaling pathway, via a PKCε-dependent mechanism, defining a Ucn2-CRFR2-PKCε-AMPK pathway that mitigates against ischemia/reperfusion injury.

Insulin-like growth factor I stimulates cardiac myosin heavy chain and actin synthesis in the awake rat

To determine the effect of insulin-like growth factor I (IGF-I) on cardiac contractile protein synthesis in vivo, we measuredl-[ ring-2,6-3H]phenylalanine incorporation into myosin heavy chain and actin during intravenous infusions (4 h) of either saline or IGF-I (1 μg ⋅ kg-1 ⋅ min-1) in awake rats. After an overnight fast, IGF-I increased myosin synthesis by 29% compared with saline (11.5 ± 0.8 vs. 8.9 ± 0.6%/day, P < 0.01) and actin synthesis by 26% (7.2 ± 0.3 vs. 5.7 ± 0.3%/day, P < 0.01), with similar effects in left and right ventricles and a comparable effect on mixed cardiac protein. When amino acids were infused with IGF-I, a further increase in myosin synthesis was observed ( P< 0.01). In fed rats, despite higher baseline synthesis rates than in fasted rats ( P < 0.01), IGF-I also increased the synthesis of myosin (12.3 ± 0.5 vs. 9.9 ± 0.5%/day, P < 0.01) and actin (8.8 ± 0.3 vs. 7.5 ± 0.2%/day, P < 0.01) compared with saline. IGF-I infusion had no hypoglycemic effect and did not change heart rate or blood pressure. Thus relatively low-dose IGF-I has a direct action in vivo to acutely increase heart contractile protein synthesis in both fasted and fed awake rats.To determine the effect of insulin-like growth factor I (IGF-I) on cardiac contractile protein synthesis in vivo, we measured L-[ring-2, 6-3H]phenylalanine incorporation into myosin heavy chain and actin during intravenous infusions (4 h) of either saline or IGF-I (1 microgram. kg-1. min-1) in awake rats. After an overnight fast, IGF-I increased myosin synthesis by 29% compared with saline (11.5 +/- 0.8 vs. 8.9 +/- 0.6%/day, P < 0.01) and actin synthesis by 26% (7.2 +/- 0.3 vs. 5.7 +/- 0.3%/day, P < 0.01), with similar effects in left and right ventricles and a comparable effect on mixed cardiac protein. When amino acids were infused with IGF-I, a further increase in myosin synthesis was observed (P < 0.01). In fed rats, despite higher baseline synthesis rates than in fasted rats (P < 0. 01), IGF-I also increased the synthesis of myosin (12.3 +/- 0.5 vs. 9.9 +/- 0.5%/day, P < 0.01) and actin (8.8 +/- 0.3 vs. 7.5 +/- 0. 2%/day, P < 0.01) compared with saline. IGF-I infusion had no hypoglycemic effect and did not change heart rate or blood pressure. Thus relatively low-dose IGF-I has a direct action in vivo to acutely increase heart contractile protein synthesis in both fasted and fed awake rats.

Modulation of Endothelial BMPR2 Activity by VEGFR3 in Pulmonary Arterial Hypertension.

Background —Bone Morphogenetic Protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP type 2 receptors (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH. Methods —We utilized a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between VEGFR3 and BMPR2. Additional in vitro studies were performed using human endothelial cells, including primary endothelial cells from subjects with PAH. Results —Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells (ECs) with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension (PH). Consistent with this data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells (PAECs) from human PAH subjects, and reconstitution of VEGFR3 expression in PAH PAECs restored BMP signaling responses. Conclusions —Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.Background: Bone morphogenetic protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP receptor type 2 (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH. Methods: We used a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between vascular endothelial growth factor receptor 3 (VEGFR3) and BMPR2. Additional in vitro studies were performed by using human endothelial cells, including primary lung endothelial cells from subjects with PAH. Results: Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial-specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension. Consistent with these data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells from human PAH subjects, and reconstitution of VEGFR3 expression in PAH pulmonary arterial endothelial cells restored BMP signaling responses. Conclusions: Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.

Restoration of Impaired Endothelial MEF2 Function Rescues Pulmonary Arterial Hypertension

Background— Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary arterioles, characterized by increased pulmonary arterial pressure and right ventricular failure. The cause of PAH is complex, but aberrant proliferation of the pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells is thought to play an important role in its pathogenesis. Understanding the mechanisms of transcriptional gene regulation involved in pulmonary vascular homeostasis can provide key insights into potential therapeutic strategies. Methods and Results— We demonstrate that the activity of the transcription factor myocyte enhancer factor 2 (MEF2) is significantly impaired in the PAECs derived from subjects with PAH. We identified MEF2 as the key cis-acting factor that regulates expression of a number of transcriptional targets involved in pulmonary vascular homeostasis, including microRNAs 424 and 503, connexins 37, and 40, and Krűppel Like Factors 2 and 4, which were found to be significantly decreased in PAH PAECs. The impaired MEF2 activity in PAH PAECs was mediated by excess nuclear accumulation of 2 class IIa histone deacetylases (HDACs) that inhibit its function, namely HDAC4 and HDAC5. Selective, pharmacological inhibition of class IIa HDACs led to restoration of MEF2 activity in PAECs, as demonstrated by increased expression of its transcriptional targets, decreased cell migration and proliferation, and rescue of experimental pulmonary hypertension models. Conclusions— Our results demonstrate that strategies to augment MEF2 activity hold potential therapeutic value in PAH. Moreover, we identify selective HDAC IIa inhibition as a viable alternative approach to avoid the potential adverse effects of broad spectrum HDAC inhibition in PAH.