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Rationale The nutrient sensing mechanistic target of rapamycin complex 1 (mTORC1) and its primary inhibitor, tuberin (TSC2), are cues for the development of cardiac hypertrophy. The phenotype of mTORC1 induced hypertrophy is unknown. Objective To examine the impact of sustained mTORC1 activation on metabolism, function, and structure of the adult heart. Methods and results We developed a mouse model of inducible, cardiac-specific sustained mTORC1 activation (mTORC1iSA) through deletion of Tsc2. Prior to hypertrophy, rates of glucose uptake and oxidation, as well as protein and enzymatic activity of glucose 6-phosphate isomerase (GPI) were decreased, while intracellular levels of glucose 6-phosphate (G6P) were increased. Subsequently, hypertrophy developed. Transcript levels of the fetal gene program and pathways of exercise-induced hypertrophy increased, while hypertrophy did not progress to heart failure. We therefore examined the hearts of wild-type mice subjected to voluntary physical activity and observed early changes in GPI, followed by hypertrophy. Rapamycin prevented these changes in both models. Conclusion Activation of mTORC1 in the adult heart triggers the development of a non-specific form of hypertrophy which is preceded by changes in cardiac glucose metabolism.

About 20-30% of cancer-associated deaths are due to complications from cachexia which is characterized by skeletal muscle atrophy. Metabolic reprogramming in cancer cells causes body-wide metabolic and proteomic remodeling, which remain poorly understood. Here, we present evidence that the oncometabolite D-2-hydroxylgutarate (D2-HG) impairs NAD+ redox homeostasis in skeletal myotubes, causing atrophy via deacetylation of LC3-II by the nuclear deacetylase Sirt1. Overexpression of p300 or silencing of Sirt1 abrogate its interaction with LC3, and subsequently reduced levels of LC3 lipidation. Using RNA-sequencing and mass spectrometry-based metabolomics and proteomics, we demonstrate that prolonged treatment with the oncometabolite D2-HG in mice promotes cachexia in vivo and increases the abundance of proteins and metabolites, which are involved in energy substrate metabolism, chromatin acetylation and autophagy regulation. We further show that D2-HG promotes a sex-dependent adaptation in skeletal muscle using network modeling and machine learning algorithms. Our multi-omics approach exposes new metabolic vulnerabilities in response to D2-HG in skeletal muscle and provides a conceptual framework for identifying therapeutic targets in cachexia.

Cancer cells rewire metabolism to support tumor growth and proliferation. In isocitrate dehydrogenase (IDH) 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including myopathy. D2-HG causes inhibition of alpha-ketoglutarate dehydrogenase (AKGDH), which is associated with reduced cardiac contractile function. How tumor cells influence the metabolism of cardiomyocytes remains mostly unknown. Specific cancer cells use glutamine-dependent reductive carboxylation to circumvent defective mitochondrial metabolism by producing citrate and acetyl-CoA for lipid synthesis, which tumors require for growth. Here, we explore the hypothesis that inhibition of AKGDH by the oncometabolite D2-HG increases glutamine-dependent reductive carboxylation in the heart. We combined ex vivo rat heart perfusions with mass-spectrometry-based stable isotope tracer studies and in silico metabolic flux analysis. In response to D2-HG-mediated inhibition of AKGDH, we observed an increased reductive carboxylation of alpha-ketoglutarate to citrate rather than oxidative decarboxylation. This pathway increases glutamine uptake and glutamine-derived citrate formation in both working rat heart perfusions and cultured adult mouse ventricular cardiomyocytes. When we perfused rat hearts with 13C-labelled D2-HG, we observed a similarly increased formation of citrate. To identify which IDH isoform is responsible for redirecting carbon flux, we modulated IDH1, 2, and 3 in adult mouse ventricular cardiomyocytes using siRNAs. Reduced expression of IDH1 impaired reductive formation of citrate. Importantly, we observed a significant correlation between reductive citrate formation and epigenetic modifications of histones, including increased histone 3 lysine 9 acetylation and di-methylation. To explore these observations, we conducted ChIP-sequencing and identified distinct transcriptional remodeling. Taken together, we demonstrate how oncometabolic stress in the heart causes redirection of central carbon metabolism via reductive carboxylation, and provide evidence of how reductive-citrate formation may induce epigenetic modifications in the heart.

Metabolic rewiring is a hallmark of cancer and muscle cells. In isocitrate dehydrogenase 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including myopathy. Our recent in vivo work showed that increased D2-HG supply by IDH-mutant cells causes heart and skeletal muscle atrophy, and decreases cellular ATP and NADH. Although heart failure and cachexia in cancer are commonly associated with chemotherapy, cancer survivors have a 5-fold increased risk of heart failure independent of any cytostatic treatment. The connection between metabolic and proteomic remodeling in this context remain poorly understood. We hypothesize that D2-HG-mediated alpha-ketoglutarate dehydrogenase (AKGDH) inhibition in myocytes results in metabolomic perturbations, increases autophagy and proteomic remodeling. Here, we report that LC3, a key regulator of autophagy, is activated in the nucleus of myocytes in presence of D2-HG through deacetylation by the nuclear deacetylase Sirt1. Activation of Sirt1 is driven by increased NAD + levels through D2-HG-mediated AKGDH inhibition. We used LC3 mutants with arginine and glutamine replacements at lysine residues to show that deacetylation of LC3 at K49 and K51 by Sirt1 shifts LC3 distribution from the nucleus into the cytosol, where it is able to undergo lipidation at pre-autophagic membranes. Live cell imaging with GFP-tagged LC3 in L6 myocytes indicated that the cycle of acetylation-deacetylation allows LC3 to redistribute from the nucleus to the cytosol within less than 24 h. Co-immunoprecipitation of LC3 followed by proteomics analysis revealed that LC3 binds to dynein in presence of D2-HG. Furthermore, D2-HG promoted skeletal muscle atrophy and reduced grip strength in wild-type C57BL/J6 mice in vivo. Using LC-MS/MS-based proteomics and metabolomics combined with RNA-sequencing, we assessed the effect of D2-HG on a systems level in skeletal muscle. Pathway-enrichment analysis revealed that D2-HG induces upregulation of key metabolic enzymes involved in glycolysis and the pentose phosphate pathway. In short, autophagy activation supports proteome remodeling in muscle cells during IDH-mutant leukemia.

The Warburg effect in cancer cells entails enhanced lactate production from glucose in the presence of oxygen. We have recently discovered footprints of the Warburg effect in the failing human heart and that metabolic remodeling precedes structural remodeling. Our aim was to test the hypothesis that

When stressed, the heart remodels metabolically and structurally. Metabolically, increases its reliance on carbohydrates for energy provision. Structurally, it hypertrophies. A common pathway for both of these changes is the activation of the mechanistic Target Of Rapamycin Complex 1 (mTORC1).

The heart remodels metabolically and structurally before it fails. Metabolically, the heart increases its reliance on carbohydrates for energy provision. Structurally, the heart hypertrophies to sustain increased hemodynamic stress. There is evidence suggesting that the activation of the mechanistic Target Of Rapamycin Complex 1 (mTORC1) pathway is closely tied to glucose uptake by the heart to drive the metabolic and structural remodeling. We have previously shown that with insulin stimulation or increases in workload, the glycolytic intermediate glucose 6-phosphate (G6P) is required to activate mTORC1. Sustained mTORC1 activation leads, in turn, to ER stress and contractile dysfunction. Studies by others in the kidney have shown that mTORC1 activation upregulates glucose transporter 1 (Glut1) expression and glucose uptake. We therefore test the hypothesis that chronic mTORC1 overactivation results in G6P accumulation, and precedes structural and functional remodeling in the heart. We developed mice with inducible, cardiac-specific deficiency of the protein tuberin (TSC2), a member of the tuberous sclerosis complex, the principal inhibitor of mTORC1. Intracellular G6P concentrations were measured enzymatically. Immunoblotting was performed on protein markers to confirm activation of mTORC1 downstream targets and of the unfolded protein response. Histologic analysis were performed to assess structural changes. Serial echocardiograms were performed to evaluate cardiac function. The results indicate that chronic mTORC1 activation through inducible, cardiac-specific deletion of TSC2 is accompanied by G6P accumulation and metabolic remodeling. Metabolic remodeling precedes structural and functional remodeling. We suggest that in the heart, sustained mTORC1 activation is a key driver of metabolic and structural remodeling.

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1 is a muscle specific ubiquitin ligase required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied. Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program were detected in both WT and Atrogin-1 -/-transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, calcinuerin, a known target of Atrogin-1 involved in hypertrophy and protein synthesis, was upregulated in unloaded Atrogin-1 deficient hearts. Consequently, μunloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. While inhibition of calcineurin decreased rates of protein synthesis in unloaded cardiomyocytes in the absence of Atrogin-1, protein synthesis rates were still higher than in WT unloaded cardiomyocytes. These results suggest that more than one pathway regulating protein synthesis is controlled by Atrogin-1 in the heart. Furthermore, the data provide evidence that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1, a muscle specific ubiquitin ligase, is required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We now tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice (8-10 weeks old, n =8-12) were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied (at least a 1.5-fold increase in heart weight, 2-fold increase in myocyte area). Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program (ANF, MHC isoform switch), were detected in both WT and Atrogin-1 -/- transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, two known targets of Atrogin-1 involved in hypertrophy and protein synthesis, calcineurin and eukaryotic initiation factor 3f, were upregulated in unloaded Atrogin-1 deficient hearts. Consequently, “unloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. The results suggest that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

Background— Autophagy is a molecular process that breaks down damaged cellular organelles and yields amino acids for de novo protein synthesis or energy provision. Mechanical unloading with a left ventricular assist device (LVAD) decreases the energy demand of the failing human heart. We tested the hypothesis that LVAD support reverses activation of autophagy. Methods and Results— Paired biopsy samples of left ventricular myocardium were obtained from 9 patients with idiopathic dilated cardiomyopathy (mean duration of LVAD support, 214 days) at the time of implantation and explantation of the LVAD. Transcript and protein levels of markers and mediators of autophagy and apoptosis were measured by quantitative reverse-transcription polymerase chain reaction and Western blotting. TUNEL assays, C9 immunohistochemistry, and 20S proteasome activity assays were also performed. Mechanical unloading significantly decreased mRNA transcript levels of Beclin-1 , autophagy-related gene 5 ( Atg5 ), and microtubule-associated protein-1 light chain-3 ( MAP1-LC3 or LC3 ; P Beclin-1 , Atg5–Atg12 conjugate, and LC3-II were also significantly reduced after LVAD support ( P Conclusions— Mechanical unloading of the failing human heart decreases markers of autophagy. These findings suggest that autophagy may be an adaptive mechanism in the failing heart, and this phenomenon is attenuated by LVAD support.

Ischemia and reperfusion (I/R) are characterized by oxidative stress as well as changes in the antioxidant enzymes of the heart. However, little is known about the transcriptional regulation of myocardial antioxidant enzymes in repetitive I/R and hibernating myocardium. In a mouse model of ischemic cardiomyopathy induced by repetitive I/R, we postulated that induction of antioxidant gene expression was dependent on reactive oxygen species (ROS). Repetitive closed-chest I/R (15 min) was performed daily in C57/BL6 mice and in mice overexpressing extracellular superoxide dismutase (EC-SOD). Antioxidant enzyme expression was measured at 3, 5, 7, and 28 days of repetitive I/R as well as 15 and 30 days after discontinuation of I/R. In order to determine whether ROS directly modulates antioxidant gene expression, transcript levels were measured in cardiomyocytes exposed to hydrogen peroxide. Repetitive I/R caused an early and sustained increase in glutathione peroxidase (GPX) transcript levels, while heme oxygenase-1 (HO-1) expression increased only after 7 days of repetitive I/R. Overexpression of EC-SOD prevented the upregulation of GPX and HO-1 transcript levels by repetitive I/R, suggesting that both genes are regulated by ROS. However, while HO-1 transcript levels increased in cardiomyocytes exposed to hydrogen peroxide, oxidative stress failed to induce the expression of GPX implying that ROS regulates GPX transcript levels only indirectly in repetitive I/R. In conclusion, repetitive I/R was associated with an early upregulation of GPX expression as well as a delayed increase of HO-1 transcript levels in the heart. The induction of both antioxidant genes was dependent on ROS, suggesting that alterations in redox balance mediate not only tissue injury but also components of "programmed cell survival" in hibernating myocardium.

Background— The peroxisome proliferators–activated receptor-α (PPARα), a transcription factor that modulates fatty acid metabolism, regulates substrate preference in the heart. Although in acute ischemia there is a switch in substrate preference from fatty acids to glucose, metabolic gene expression in repetitive ischemia is not well described. In a mouse model of ischemic cardiomyopathy induced by repetitive ischemia/reperfusion (I/R), we postulated that downregulation of PPARα is regulated by reactive oxygen species and is necessary for maintaining contractile function in the heart. Methods and Results— Repetitive closed-chest I/R (15 minutes) was performed daily in C57/BL6 mice, mice overexpressing extracellular superoxide dismutase, and mice treated with the PPARα agonist-WY-14,643. Echocardiography, histology, and candidate gene expression were measured at 3, 5, 7, and 28 days of repetitive I/R and 15 and 30 days after discontinuation of I/R. Repetitive I/R was associated with a downregulation of PPARα-regulated genes and both myosin heavy chain isoform transcript levels, which was reversible on discontinuation of I/R. Overexpression of EC-SOD prevented the downregulation of PPARα-regulated genes and myosin iso-genes by repetitive I/R. Furthermore, reactivation of PPARα in mice exposed to repetitive I/R worsened contractile function, induced microinfarctions, and increased intramyocardial triglyceride deposition, features suggestive of cardiac lipotoxicity. Conclusions— Metabolic and myosin isoform gene expression in repetitive I/R is mediated by reactive oxygen species. Furthermore, we suggest that downregulation of PPARα in repetitive I/R is an adaptive mechanism that is able to prevent lipotoxicity in the ischemic myocardium.

Introduction: Ranolazine, a drug believed to partially inhibit the oxidation of long-chain fatty acids, has previously been shown to increase treadmill exercise performance in patients with ischemic heart disease. The drug’s long-term efficacy and safety have not been studied in a more realistic setting—that is, in combination with β-blockers or calcium antagonists in patients with chronic, severe angina.