SUMMARY Remodeling of the tricarboxylic acid (TCA) cycle is a metabolic adaptation accompanying inflammatory macrophage activation. During this process, endogenous metabolites can adopt regulatory roles that govern specific aspects of inflammatory response, as recently shown for succinate, which regulates the pro-inflammatory IL-1β-HIF-1α axis. Itaconate is one of the most highly induced metabolites in activated macrophages, yet its functional significance remains unknown. Here, we show that itaconate modulates macrophage metabolism and effector functions by inhibiting succinate dehydrogenase-mediated oxidation of succinate. Through this action, itaconate exerts anti-inflammatory effects when administered in vitro and in vivo during macrophage activation and ischemia-reperfusion injury. Using newly generated Irg1−/− mice, which lack the ability to produce itaconate, we show that endogenous itaconate regulates succinate levels and function, mitochondrial respiration, and inflammatory cytokine production during macrophage activation. These studies highlight itaconate as a major physiological regulator of the global metabolic rewiring and effector functions of inflammatory macrophages.

We hypothesized that the lipid-activated transcription factor, the peroxisome proliferator-activated receptor ␣ (PPAR␣), plays a pivotal role in the cellular metabolic response to fasting. Short-term starvation caused hepatic steatosis, myocardial lipid accumulation, and hypoglycemia, with an inadequate ketogenic response in adult mice lacking PPAR␣ (PPAR␣ ؊͞؊ ), a phenotype that bears remarkable similarity to that of humans with genetic defects in mitochondrial fatty acid oxidation enzymes. In PPAR␣ ؉͞؉ mice, fasting induced the hepatic and cardiac expression of PPAR␣ target genes encoding key mitochondrial (mediumchain acyl-CoA dehydrogenase, carnitine palmitoyltransferase I) and extramitochondrial (acyl-CoA oxidase, cytochrome P450 4A3) enzymes. In striking contrast, the hepatic and cardiac expression of most PPAR␣ target genes was not induced by fasting in PPAR␣ ؊͞؊ mice. These results define a critical role for PPAR␣ in a transcriptional regulatory response to fasting and identify the PPAR␣ ؊͞؊ mouse as a potentially useful murine model of inborn and acquired abnormalities of human fatty acid utilization.Starvation triggers a complex array of adaptive metabolic responses. A prominent feature of the energy-metabolic response to fasting includes a switch to reliance on fatty acids and ketones for energy production (1-4) and an augmentation in the capacity for mitochondrial fatty acid oxidation (FAO) in tissues with high oxidative energy demands such as heart and liver (5). The importance of the fasting-inducible capacity for cellular lipid utilization is underscored by the dramatic phenotype of human inborn errors in mitochondrial FAO enzymes (6). Children afflicted with genetically determined enzymatic defects in the FAO pathway typically are asymptomatic under normal feeding conditions. However, short-term fasting, such as that associated with an infectious illness, precipitates a dramatic and often fatal clinical picture characterized by hypoketotic hypoglycemia, liver dysfunction, and cardiomyopathy (6-8). Postmortem studies of FAO enzyme-deficient patients have demonstrated marked intracellular accumulation of neutral lipid in liver and heart. The capacity to oxidize fats is also diminished in several common acquired cardiac diseases including cardiac hypertrophy and myocardial ischemia (9-17). The molecular pathogenesis of target organ dysfunction from inherited and acquired alterations in cellular FAO has not been elucidated.A previous study in rodents demonstrated that the hepatic expression of genes encoding mitochondrial FAO enzymes is induced, at the transcriptional level, in response to fasting (5). This transcriptional regulatory response likely plays a key role in the fasting-induced augmentation of FAO capacity in liver and other oxidative tissues. The mechanisms involved in the fasting-induced transcriptional activation of FAO enzyme genes are unknown. However, recent studies have identified a role for a nuclear receptor, the peroxisome proliferatoractivated receptor ␣ (PPAR␣), in the ...

Background In myocardial ischemia, induction of autophagy via the AMP-induced protein kinase (AMPK) pathway is protective, whereas reperfusion stimulates autophagy with BECLIN-1 upregulation, and is implicated in causing cell death. We examined flux through the macro-autophagy pathway as a determinant of the discrepant outcomes in cardiomyocyte cell death in this setting Methods and Results Reversible left anterior descending coronary artery ligation was performed in mice with cardiomyocyte-restricted expression of GFP-tagged microtubule associated protein light chain-3 (LC3) to induce ischemia (120 minutes) or ischemia-reperfusion (IR, 30–90 minutes) with saline or chloroquine (CQ) pretreatment (n=4/group). Autophagosome clearance, assessed as the ratio of punctate LC3 abundance in saline to CQ treated samples was markedly impaired with IR as compared with sham controls. Reoxygenation increased cell death in neonatal rat cardiomyocytes (NRCMs) as compared with hypoxia alone; markedly increased autophagosomes but not autolysosomes (assessed as punctate dual fluorescent mCherry-GFP tandem tagged LC3 expression); and impaired clearance of polyglutamine aggregates, indicating impaired autophagic flux. The resultant autophagosome accumulation was associated with increased reactive oxygen species (ROS) and mitochondrial permeabilization leading to cell death, which was attenuated by cyclosporine A pretreatment. Hypoxia-reoxygenation injury was accompanied by ROS-mediated BECLIN-1 upregulation and reduction in Lysosome Associated Membrane Protein-2 (LAMP2), a critical determinant of autophagosome-lysosome fusion. Restoration of LAMP2 levels synergizes with partial BECLIN-1 knockdown to restore autophagosome processing and attenuate cell death following hypoxia-reoxygenation. Conclusions Ischemia-reperfusion injury impairs autophagosome clearance mediated in part by ROS-induced decline in LAMP2 and upregulation of BECLIN-1, contributing to increased cardiomyocyte death.

Background-Postnatal growth of the heart chiefly involves nonproliferative cardiomyocyte enlargement. Cardiac hypertrophy exists in a "physiological" form that is an adaptive response to long-term exercise training and as a "pathological" form that often is a maladaptive response to provocative stimuli such as hypertension and aortic valvular stenosis. A signaling cascade that includes the protein kinase Akt regulates the growth and survival of many cell types, but the precise role of Akt1 in either form of cardiac hypertrophy is unknown. Methods and Results-To evaluate the role of Akt1 in physiological cardiac growth, akt1 Ϫ/Ϫ adult murine cardiac myocytes (AMCMs) were treated with IGF-1, and akt1 Ϫ/Ϫ mice were subjected to exercise training. akt1 Ϫ/Ϫ AMCMs were resistant to insulin-like growth factor-1-stimulated protein synthesis. The akt1 Ϫ/Ϫ mice were found to be resistant to swimming training-induced cardiac hypertrophy. To evaluate the role of Akt in pathological cardiac growth, akt1

Rationale-Recent advancements have brought to light the origins, complexity, and functions of tissue-resident macrophages. However, in the context of tissue injury or disease, large numbers of monocytes infiltrate the heart and are thought to contribute to adverse remodeling and heart failure pathogenesis. Little is understood about the diversity of monocytes and monocyte-derived macrophages recruited to the heart after myocardial injury, including the mechanisms that regulate monocyte recruitment and fate specification.Objective-We sought to test the hypothesis that distinct subsets of tissue-resident CCR2− (C-C chemokine receptor 2) and CCR2+ macrophages orchestrate monocyte recruitment and fate specification after myocardial injury.Methods and Results-We reveal that in numerous mouse models of cardiomyocyte cell death (permanent myocardial infarction, reperfused myocardial infarction, and diphtheria toxin cardiomyocyte ablation), there is a shift in macrophage ontogeny whereby tissue-resident macrophages are predominately replaced by infiltrating monocytes and monocyte-derived macrophages. Using syngeneic cardiac transplantation to model ischemia-reperfusion injury and distinguish tissue-resident from recruited cell populations in combination with intravital 2-photon microscopy, we demonstrate that monocyte recruitment is differentially orchestrated by distinct subsets of tissue-resident cardiac macrophages. Tissue-resident CCR2+ macrophages promote monocyte recruitment through an MYD88 (myeloid differentiation primary response 88)dependent mechanism that results in release of MCPs (monocyte chemoattractant proteins) and monocyte mobilization. In contrast, tissue-resident CCR2− macrophages inhibit monocyte recruitment. Using CD (cluster of differentiation) 169-DTR (diphtheria toxin receptor) and CCR2-DTR mice, we further show that selective depletion of either tissue-resident CCR2− or CCR2+ macrophages before myocardial infarction results in divergent effects on left ventricular function, Bajpai et al.

IntroductionMyocardial energy substrate preference is tightly controlled in mammalian organisms during development and in response to diverse dietary, physiologic, and pathophysiologic conditions (1, 2). During the fetal period, glucose and lactate serve as the chief myocardial energy substrates. After birth and during the postnatal period, myocardial energy is derived increasingly from reducing equivalents generated by mitochondrial β-oxidation of long-chain fatty acids (3). In the normal adult heart, mitochondrial fatty acid oxidation (FAO) accounts for the majority of ATP production (1). The importance of the FAO pathway as a source of energy in the postnatal human heart is underscored by the severe clinical manifestations of genetic defects in mitochondrial FAO enzymes, including childhood cardiomyopathy and sudden death, presumably due to the accumulation of myocardial long-chain fatty acid intermediates coupled with depletion of energy stores (4).The results of studies performed in cell culture and in vivo have established a critical role for members of the nuclear receptor superfamily in the transcriptional control of genes encoding cardiac FAO enzymes (5-7).Peroxisome proliferator-activated receptor-α (PPARα), a lipid-activated nuclear receptor (8), has been shown to regulate basal and fatty acid-induced transcription of FAO enzyme genes, including medium-chain acylCoA dehydrogenase (5) and muscle carnitine palmitoyltransferase I (M-CPT I or CPT Iβ) (9, 10). PPARα binds to target DNA elements as a heterodimeric partner with the retinoid X receptor, and is activated by a variety of ligands, including long-chain fatty acids (11). The expression of mitochondrial and peroxisomal FAO enzymes are reduced in postnatal liver and heart of PPARα-null (PPARα -/-) mice (12, 13). Moreover, PPARα -/-mice accumulate myocardial lipid in the context of conditions known to increase FAO rates, such as fasting, indicating that PPARα plays a critical role in the maintenance of cardiac energy and lipid homeostasis by its regulatory influence on cellular fatty acid utilization pathways (14,15).During the development of pressure overloadinduced ventricular hypertrophy, myocardial FAO rates decrease and glucose utilization increases, a reversion to the fetal pattern of energy substrate utilization (16-18). The expression of mitochondrial We sought to delineate the molecular regulatory events involved in the energy substrate preference switch from fatty acids to glucose during cardiac hypertrophic growth. α 1 -adrenergic agonist-induced hypertrophy of cardiac myocytes in culture resulted in a significant decrease in palmitate oxidation rates and a reduction in the expression of the gene encoding muscle carnitine palmitoyltransferase I (M-CPT I), an enzyme involved in mitochondrial fatty acid uptake. Cardiac myocyte transfection studies demonstrated that M-CPT I promoter activity is repressed during cardiac myocyte hypertrophic growth, an effect that mapped to a peroxisome proliferator-activated receptor-α (PPARα) response element...

Abstract-Evidence is emerging that systemic metabolic disturbances contribute to cardiac myocyte dysfunction and clinically apparent heart failure, independent of associated coronary artery disease. To test the hypothesis that perturbation of lipid homeostasis in cardiomyocytes contributes to cardiac dysfunction, we engineered transgenic mice with cardiac-specific overexpression of fatty acid transport protein 1 (FATP1) using the ␣-myosin heavy chain gene promoter. Two independent transgenic lines demonstrate 4-fold increased myocardial free fatty acid (FFA) uptake that is consistent with the known function of FATP1. Increased FFA uptake in this model likely contributes to early cardiomyocyte FFA accumulation (2-fold increased) and subsequent increased cardiac FFA metabolism (2-fold). By 3 months of age, transgenic mice have echocardiographic evidence of impaired left ventricular filling and biatrial enlargement, but preserved systolic function. Doppler tissue imaging and hemodynamic studies confirm that these mice have predominantly diastolic dysfunction. Furthermore, ambulatory ECG monitoring reveals prolonged QT c intervals, reflecting reductions in the densities of repolarizing, voltage-gated K ϩ currents in ventricular myocytes. Our results show that in the absence of systemic metabolic disturbances, such as diabetes or hyperlipidemia, perturbation of cardiomyocyte lipid homeostasis leads to cardiac dysfunction with pathophysiological findings similar to those in diabetic cardiomyopathy. Moreover, the MHC-FATP model supports a role for FATPs in FFA import into the heart in vivo. Key Words: lipids Ⅲ metabolism Ⅲ cardiomyopathy C ardiomyopathy has been observed in a variety of metabolic disorders. In inherited disorders of ␤-oxidation, accumulation of unmetabolized lipid in cardiac myocytes is associated with ventricular systolic dysfunction. 1 In obesity, increased myocardial oxygen consumption and decreased efficiency may contribute to diastolic and systolic dysfunction. 2,3 In diabetes mellitus, heart failure in the absence of valvular or congenital heart disease, alcoholism, hypertension, or significant epicardial coronary atherosclerosis is defined as diabetic cardiomyopathy and accounts for significant morbidity and mortality in people with type 1 and type 2 diabetes. 4 Echocardiographic and hemodynamic studies suggest left ventricular (LV) diastolic impairment represents an early preclinical manifestation of diabetic cardiomyopathy that may progress over an extended period of time to both diastolic and systolic dysfunction. 5,6 In these metabolic disorders, systemic metabolic perturbations lead to myocyte dysfunction and/or loss. Glucotoxicity, 7 ATP depletion, 8 and maladaptive changes in metabolic substrate utilization 9 are mechanisms proposed to contribute to cardiac dysfunction. It has also been hypothesized that mismatch between tissue free fatty acid (FFA) import and utilization leads to lipid accumulation and results in lipotoxicity. In diabetes, this imbalance results from high-serum F...

The peroxisome proliferator-activated receptor ␣ (PPAR ␣ ) is a nuclear receptor implicated in the control of cellular lipid utilization. To test the hypothesis that PPAR ␣ is activated as a component of the cellular lipid homeostatic response, the expression of PPAR ␣ target genes was characterized in response to a perturbation in cellular lipid oxidative flux caused by pharmacologic inhibition of mitochondrial fatty acid import. Inhibition of fatty acid oxidative flux caused a feedback induction of PPAR ␣ target genes encoding fatty acid oxidation enzymes in liver and heart. In mice lacking PPAR ␣ (PPAR ␣Ϫ / Ϫ ), inhibition of cellular fatty acid flux caused massive hepatic and cardiac lipid accumulation, hypoglycemia, and death in 100% of male, but only 25% of female PPAR ␣Ϫ / Ϫ mice. The metabolic phenotype of male PPAR ␣Ϫ / Ϫ mice was rescued by a 2-wk pretreatment with ␤ -estradiol. These results demonstrate a pivotal role for PPAR ␣ in lipid and glucose homeostasis in vivo and implicate estrogen signaling pathways in the regulation of cardiac and hepatic lipid metabolism. ( J.