Dake Qi

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.

A small molecule AMPK activator protects the heart against ischemia-reperfusion injury

AMP-activated protein kinase (AMPK) is a stress signaling enzyme that orchestrates the regulation of energy-generating and -consuming pathways. Intrinsic AMPK activation protects the heart against ischemic injury and apoptosis, but whether pharmacologic AMPK stimulation mitigates ischemia-reperfusion damage is unknown. The aims of this study were to determine whether direct stimulation of AMPK using a small molecule activator, A-769662, attenuates myocardial ischemia-reperfusion injury and to examine its cardioprotective mechanisms. Isolated mouse hearts pre-treated with A-769662 had better recovery of left ventricular contractile function (55% vs. 29% of baseline rate-pressure product; p=0.03) and less myocardial necrosis (56% reduction in infarct size; p<0.01) during post-ischemic reperfusion compared to control hearts. Pre-treatment with A-769662 in vivo attenuated infarct size in C57Bl/6 mice undergoing left coronary artery occlusion and reperfusion compared to vehicle (36% vs. 18%, p=0.025). Mouse hearts with genetically inactivated AMPK were not protected by A-769662, indicating the specificity of this compound. Pre-treatment with A-769662 increased the phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2), preserved energy charge during ischemia, delayed the development of ischemic contracture, and reduced myocardial apoptosis and necrosis. A-769662 also augmented endothelial nitric oxide synthase (eNOS) activation during ischemia, which partially attenuated myocardial stunning, but did not prevent necrosis. AMPK is a therapeutic target that can be stimulated by a direct-acting small molecule in order to prevent injury during ischemia-reperfusion. The use of AMPK activators may represent a novel strategy to protect the heart and other solid organs against ischemia.

AMPK: energy sensor and survival mechanism in the ischemic heart

AMP-activated protein kinase (AMPK) is a critical regulator of cellular metabolism and plays an important role in diabetes, cancer, and vascular disease. In the heart, AMPK activation is an essential component of the adaptive response to cardiomyocyte stress that occurs during myocardial ischemia. During ischemia-reperfusion, AMPK activation modulates glucose and fatty acid metabolism, mitochondrial function, endoplasmic reticulum (ER) stress, autophagy, and apoptosis. Pharmacological activation of AMPK prevents myocardial necrosis and contractile dysfunction during ischemia-reperfusion and potentially represents a cardioprotective strategy for the treatment of myocardial infarction. This review discusses novel mechanisms of AMPK activation in the ischemic heart, the role of endogenous AMPK activation during ischemia, and the potential therapeutic applications for AMPK-directed therapy.

TLR4 regulates cardiac lipid accumulation and diabetic heart disease in the nonobese diabetic mouse model of type 1 diabetes

Toll-like receptor (TLR)4 regulates inflammation and metabolism and has been linked to the pathogenesis of heart disease. TLR4 is upregulated in diabetic cardiomyocytes, and we examined the role of TLR4 in modulating cardiac fatty acid (FA) metabolism and the pathogenesis of diabetic heart disease in nonobese diabetic (NOD) mice. Both wild-type (WT) NOD and TLR4-deficient NOD animals had increased plasma triglyceride levels after the onset of diabetes. However, by comparison, TLR4-deficient NOD mouse hearts had lower triglyceride accumulation in the early stages of diabetes, which was associated with a reduction in myeloid differentiation primary response gene (88) (MyD88), phosphorylation of p38 MAPK (phospho-p38), lipoprotein lipase (LPL), and JNK levels but increased phospho-AMP-activated protein kinase (AMPK). Oleic acid treatment in H9C2 cardiomyocytes also led to cellular lipid accumulation, which was attenuated by TLR4 small interfering RNA. TLR4 deficiency in the cells decreased FA-induced augmentation of MyD88, phospho-p38, and LPL, suggesting that TLR4 may modulate FA-induced lipid metabolism in cardiomyocytes. In addition, although cardiac function was impaired in both diabetic WT NOD and TLR4-deficient NOD animals compared with control nondiabetic mice, this deficit was less in the diabetic TLR4-deficient NOD mice, which had greater ejection fraction, greater fractional shortening, and increased left ventricular developed pressure in the early stages after the development of diabetes compared with their diabetic WT NOD counterparts. Thus, we conclude that TLR4 plays a role in regulating lipid accumulation in cardiac muscle after the onset of type 1 diabetes, which may contribute to cardiac dysfunction.

Circulating triglyceride lipolysis facilitates lipoprotein lipase translocation from cardiomyocyte to myocardial endothelial lining

OBJECTIVE Lipoprotein lipase (LPL) mediated hydrolysis of circulating triglyceride (TG)-rich lipoproteins provides the heart with fatty acids. The present study was designed to investigate the influence of circulating TG and their lipolysis in facilitating translocation of LPL from the underlying cardiomyocyte cell surface to the coronary lumen. METHODS The in vivo effects of diazoxide (DZ), an agent that causes rapid hypoinsulinemia, and the in vitro effect of the lipoprotein breakdown product L-alpha-lysophosphatidylcholine (Lyso-PC) on luminal LPL were examined in Wistar rats. Manipulation of circulating TG in DZ-treated animals and their influence on LPL was also determined. RESULTS Within 4 h following DZ a major increase in LPL activity and protein occurred at the coronary lumen. Myocyte cell surface LPL was reduced 50% subsequent to DZ. Exposure of isolated control hearts to 1 nM Lyso-PC enhanced luminal LPL to levels observed following DZ. Treatment of DZ animals with either WR 1339 (inhibits circulating TG breakdown) or N(6)-cyclopentyladenosine (inhibits adipose tissue lipolysis) decreased DZ induced augmentation of cardiac LPL. CONCLUSIONS Using DZ, our studies for the first time demonstrate that LPL at the coronary lumen can be augmented as early as 4 h after hypoinsulinemia and that this increase likely involves posttranslational processing via TG breakdown of circulating lipoproteins and a Lyso-PC dependent mechanism.

Inflammatory Regulation by TLR3 in Acute Hepatitis

TLR3 is known to respond to dsRNA from viruses, apoptotic cells, and/or necrotic cells. Dying cells are a rich source of ligands that can activate TLRs, such as TLR3. TLR3 expressed in the liver is likely to be a mediator of innate activation and inflammation in the liver. The importance of this function of TLR3 during acute hepatitis has not previously been fully explored. We used the mouse model of Con A-induced hepatitis and observed a novel role for TLR3 in hepatocyte damage in the absence of an exogenous viral stimulus. Interestingly, TLR3 expression in liver mononuclear cells and sinus endothelial cells was up-regulated after Con A injection and TLR3−/− mice were protected from Con A-induced hepatitis. Moreover, splenocytes from TLR3−/− mice proliferated less to Con A stimulation in the presence of RNA derived from damaged liver tissue compared with wild-type (WT) mice. To determine the relative contribution of TLR3 expression by hematopoietic cells or nonhematopoietic to liver damage during Con A-induced hepatitis, we generated bone marrow chimeric mice. TLR3−/− mice engrafted with WT hematopoietic cells were protected in a similar manner to WT mice reconstituted with TLR3−/− bone marrow, indicating that TLR3 signaling in both nonhematopoietic and hematopoietic cells plays an important role in mediating liver damage. In summary, our data suggest that TLR3 signaling is necessary for Con A-induced liver damage in vivo and that TLR3 regulates inflammation and the adaptive T cell immune response in the absence of viral infection.

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.

Venlafaxine inhibits the upregulation of plasma tumor necrosis factor-alpha (TNF-α) in the Chinese patients with major depressive disorder: A prospective longitudinal study

Although tumor necrosis factor-alpha (TNF-α) has been recognized to be involved in the pathogenesis of major depressive disorder (MDD) for a long time, only few studies so far investigated the effects of antidepressant, venlafaxine on TNF-α and the results are inconsistent. Moreover, the association between plasma TNF-α levels and suicide accompanied with MDD is entirely unknown. To elucidate these relationships, in the present study, 64 first-episode drug-naïve MDD patients and 64 matched healthy controls were recruited. Total 61 MDD patients received 8-week venlafaxine treatment and they were divided into responders and non-responders according to the reduction rate of HRSD-17. Prior to venlafaxine treatment, both responders and non-responders shared a similar plasma TNF-α (p=0.33), which was significantly decreased following venlafaxine treatment (p<0.001, p=0.03, respectively). Compared to non-responders, the responder group had a greater reduction in TNF-α (p=0.01), which was associated with the greater reduction rate of HRSD-17 (B=1.02, p=0.01). In addition, the plasma TNF-α levels were equally higher in both suicidal and non-suicidal MDD patients (p=0.84) compared to the healthy controls on admission (p=0.001, p=0.03, respectively). Together, our data suggest that MDD per se rather than suicide is associated with the elevated plasma TNF-α, which can be inhibited with venlfaxine monotherapy. The extent of TNF-α reduction may be associated with the efficiency of venlafaxine.

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.