Metabolites In Heart Research Articles

Mammalian Diaphanous-related formin 1 (mDia1) inducing diabetic cardiomyopathy by regulating metabolic reprogramming

Abstract Background Mammalian Diaphanous-related formin 1 (mDia1) is one of the formin family proteins and acts as a rho-effector molecule which is involved in actin organization. Previous studies showed that mDia1 aggravated compensatory cardiac hypertrophy in response to pressure overload. However, the role of mDia1 in diabetic cardiomyopathy and its mechanisms remain elusive. Objective To clarify the mechanisms of mDia1 in DCM through mediating metabolic reprogramming. Methods 5-week-old of mDia1 ko mice were used to induce DCM by feeding high-fat diet (HFD) for 16 weeks. The blood glucose and body weight of the mice were measured regularly. Echocardiography was used to evaluate cardiac function. HE and WGA staining were used to evaluate hypertrophy of cardiomyocytes. Transmission electron microscopy and mitotracker staining were used to observe the morphology of mitochondrial in heart. Proteomics and metabolomics were used to explore the metabolites in hearts of mice. Primary neonatal cardiomyocytes were isolated and stimulated with 35 mM glucose and 250 μM palmitate in vitro. Adenovirus expressing shRNA of mDia1 was used to knockdown mDia1 in cardiomyocytes. Mitochondria were isolated from primary cardiomyocytes to verify the translocation of mDia1. Co-Immunoprecipitation was used to confirm the interaction between mDia1 with succinate dehydrogenase subunits A (SDHA). Results mDia1 had no effects to blood glucose and body weight after 16-week HFD feeding. Loss of mDia1 ameliorated diastolic dysfunction in DCM mice with shorter isovolumic relaxation time (IVRT) and increased E/A ratio in left ventricular. In addition, cardiac hypertrophy was significantly improved in mDia1 ko mice. mDia1 deficiency tended to maintain the normal mitochondrial structure. Results of proteomics showed that mDia1 was involved in mitochondrial function, ATP synthesis and metabolic pathways. Metabolomics showed that HFD induced accumulation of α-ketoglutaric acid in wild type mice compared with mDia1 ko mice. However, mDia1 ko significantly increased fumaric acid but did not affect succinyl-CoA and succinic acid compared with wild type mice. Furthermore，we found that mDia1 translocated into mitochondrial with high glucose and palmitate stimuli. Results of CoIP showed mDia1 interacted with SDHA both in cardiomyocytes and HEK293 cells. Conclusions mDia1 deteriorated DCM through translocating into mitochondrial and interacting with SDHA, a process dependent on metabolic pathways.Figure of Mechanism

Abstract MP58: Tissue And Sex Specific Effects Of Gstm1 Deletion In Mouse Models

The glutathione S-transferase ( Gst ) gene family encodes antioxidant enzymes. In humans, a common null allele deletion variant of GST μ-1 ( GSTM1 ) is highly prevalent across populations and is associated with increased risk and progression of various diseases. Using a Gstm1 knockout (KO) mouse model, we previously showed that KO mice with angiotensin II-induced hypertension (HTN) have increased kidney injury compared to wild-type (WT) controls, mediated by elevated oxidative stress. In the same mouse model, we have recently reported that in a Langendorff-perfused cardiac ischemia-reperfusion injury (IRI) model, where damage is also mediated by oxidative stress, male KO hearts are protected while females are not. Here, we investigated the molecular mechanisms for this difference in male hearts. WT and KO mice of both sexes were studied at 12-20 weeks of age. Hearts were snap frozen at baseline and after 25 min of global ischemia, and kidneys were collected at baseline and 4 weeks following HTN induction. A panel of 18 Gst genes were probed by qPCR from baseline hearts and kidneys of both sexes. Global metabolites were assayed using Metabolon, Inc. from hearts of both sexes and kidneys of males, at both baseline and diseased states. Analysis by qPCR (n = 3/group) showed that male, but not female, KO hearts had upregulation of other Gst s. In contrast, no significant differences between were found in male kidneys. Metabolomics (n = 6/group) detected 695 metabolites in hearts and 926 in kidneys. There were increases in several metabolites in KO vs. WT hearts including those with antioxidant properties. Notably, increases in carnosine and anserine were observed in KO male hearts but not in female hearts, while that of other antioxidant-related metabolites were observed in hearts of both sexes, but not in kidneys. HTN induced significant increases in metabolites in KO vs. WT kidneys in the pathways related to and linking methionine, cysteine, and glutathione, which were not observed in hearts. In this study, gene expression and metabolites suggest that the mechanisms compensating for the loss of GSTM1 are both tissue and sex specific. The resulting differences in antioxidant enzymes and metabolites may explain the unexpected protection for male Gstm1 KO hearts in IRI.

Effect of dietary sulphur amino acid levels and guanidinoacetic acid supplementation on performance, carcase yield and energetic molecular metabolites in broiler chickens fed wheat-soy diets

The aim of the present study was to evaluate the effects of dietary total sulphur amino acids (TSAA) levels and guanidinoacetic acid (GAA) supplementation on performance, carcase yield and energetic molecular metabolites in heart and breast muscle of broiler chickens fed wheat-soy diet. A total of 450, male one-day-old Ross-308 broiler chicks were assigned in a 3 × 3 factorial arrangement of nine treatments, five replicates and 10 birds each. Dietary treatments included three graded levels of supplementary TSAA (+0.4 g/kg per level) resulting in a deficient, adequate and plus level of recommended by strain, with three levels of GAA (0, 0.6 and 1.2 g/kg) and were fed for 35 days. Dietary TSAA levels only enhanced performance during the starter period (p < .05). At adequate TSAA level, weight gain (WG) and feed conversion ratio (FCR) in the grower phase were positively affected by supplementing GAA at 1.2 g/kg (p < .05). The FCR during the overall experimental period was significantly lower when feeding GAA at 0.6 and 1.2 g/kg compared to non-supplemented (p < .05). Heart and breast muscle ATP/AMP ratio and breast muscle ADP and PCr concentration and PCr/CrN ratio in the birds fed 1.2 g/kg GAA diet were significantly higher than non-supplemented (p < .05). It is concluded, effects of dietary GAA supplementation were influenced by dietary TSAA level, this indicates to need for proper dietary sulphur amino acid formulation by GAA supplementation. Dietary GAA supplementation could improve broiler chicken’s growth performance via regulating some aspects of energy metabolism. HIGHLIGHTS Guanidinoacetic acid plays a critical role in energy homeostasis. The effect of dietary Guanidinoacetic acid supplementation may influenced by the dietary sulphur amino acids.

Dietary branched-chain amino acid restriction alters fuel selection and reduces triglyceride stores in hearts of Zucker fatty rats.

Elevations in circulating levels of branched-chain amino acids (BCAAs) are associated with a variety of cardiometabolic diseases and conditions. Restriction of dietary BCAAs in rodent models of obesity lowers circulating BCAA levels and improves whole-animal and skeletal-muscle insulin sensitivity and lipid homeostasis, but the impact of BCAA supply on heart metabolism has not been studied. Here, we report that feeding a BCAA-restricted chow diet to Zucker fatty rats (ZFRs) causes a shift in cardiac fuel metabolism that favors fatty acid relative to glucose catabolism. This is illustrated by an increase in labeling of acetyl-CoA from [1-13C]palmitate and a decrease in labeling of acetyl-CoA and malonyl-CoA from [U-13C]glucose, accompanied by a decrease in cardiac hexokinase II and glucose transporter 4 protein levels. Metabolomic profiling of heart tissue supports these findings by demonstrating an increase in levels of a host of fatty-acid-derived metabolites in hearts from ZFRs and Zucker lean rats (ZLRs) fed the BCAA-restricted diet. In addition, the twofold increase in cardiac triglyceride stores in ZFRs compared with ZLRs fed on chow diet is eliminated in ZFRs fed on the BCAA-restricted diet. Finally, the enzymatic activity of branched-chain ketoacid dehydrogenase (BCKDH) is not influenced by BCAA restriction, and levels of BCAA in the heart instead reflect their levels in circulation. In summary, reducing BCAA supply in obesity improves cardiac metabolic health by a mechanism independent of alterations in BCKDH activity.

Metabolic adaptations during extreme anoxia in the turtle heart and their implications for ischemia-reperfusion injury

ATP depletion and succinate accumulation during ischemia lead to oxidative damage to mammalian organs upon reperfusion. In contrast, freshwater turtles survive weeks of anoxia at low temperatures without suffering from oxidative damage upon reoxygenation, but the mechanisms are unclear. To determine how turtles survive prolonged anoxia, we measured ~80 metabolites in hearts from cold-acclimated (5 °C) turtles exposed to 9 days anoxia and compared the results with those for normoxic turtles (25 °C) and mouse hearts exposed to 30 min of ischemia. In turtles, ATP and ADP decreased to new steady-state levels during fasting and cold-acclimation and further with anoxia, but disappeared within 30 min of ischemia in mouse hearts. High NADH/NAD+ ratios were associated with succinate accumulation in both anoxic turtles and ischemic mouse hearts. However, succinate concentrations and succinate/fumarate ratios were lower in turtle than in mouse heart, limiting the driving force for production of reactive oxygen species (ROS) upon reoxygenation in turtles. Furthermore, we show production of ROS from succinate is prevented by re-synthesis of ATP from ADP. Thus, maintenance of an ATP/ADP pool and low succinate accumulation likely protects turtle hearts from anoxia/reoxygenation injury and suggests metabolic interventions as a therapeutic approach to limit ischemia/reperfusion injury in mammals.

Abstract 296: Cd38 Deficiency Protects Heart From Lipid Overload-induced Oxidative Stress via Activating Sirt3/foxo3 Pathway

Background/Aims: Our and other studies showed that CD38 deficiency protected heart from ischemia/reperfusion injury and high fat diet (HFD)-induced obesity in mice, respectively. However, the roles of CD38 in lipid overload-induced heart injury were not evaluated. In the present study, we investigated the preventive effects and mechanisms of CD38 deficiency on mouse heart injury induced by HFD. Methods: The discrepant metabolites in heart from wild type (WT) and CD38 knockout (CD38 -/- ) mice fed with HFD were examined using metabolomics analysis. The cell viability, the lactate hydrogenase (LDH) release, super oxide dismutase (SOD) activity, reactive oxygen species (ROS) production, triglyceride concentration and the expressions of various corresponding genes were examined for evaluating the effects of CD38 on oleic acid (OA)-induced cell injury using CD38 knockdown H9C2 stable cell line. Results: Our results revealed that CD38 deficiency significantly elevated the intracellular glutathione (GSH) concentration and GSH/GSSG ratio, decreased the contents of various free fatty acids and increased intracellular NAD + level in heart of CD38 -/- mice fed with HFD. Knockdown of CD38 significantly attenuated OA-induced cell injury, ROS production and lipid synthesis, seen as enhancing cell viability, reducing LDH release, elevating SOD activity, and decreasing triglyceride content and expressions of FASN in the CD38 knockdown H9C2 stable cell line. In addition, we also observed that the mitochondrial deacetylase Sirt3 and its target genes FOXO3 and SOD2 were upregulated in the stable cell lines after OA stimulation. Furthermore, the expressions of NOX2 and NOX4 were attenuated, accompanied by decreasing Bax expression and increasing Bcl-2 expression in the cells after OA stimulation. Conclusions: Our results demonstrated that CD38 deficiency protected heart from HFD-induced oxidative stress via activating Sirt3/FOXO3-mediated anti-oxidative stress pathway.

CD38 Deficiency Protects Heart from High Fat Diet-Induced Oxidative Stress Via Activating Sirt3/FOXO3 Pathway

Background/Aims: Previous studies showed that CD38 deficiency protected heart from ischemia/reperfusion injury and high fat diet (HFD)-induced obesity in mice. However, the role of CD38 in HFD-induced heart injury remains unclear. In the present study, we have investigated the effects and mechanisms of CD38 deficiency on HFD-induced heart injury. Methods: The metabolites in heart from wild type (WT) and CD38 knockout (CD38^-/-) mice were examined using metabolomics analysis. Cell viability, lactate hydrogenase (LDH) release, super oxide dismutase (SOD) activity, reactive oxygen species (ROS) production, triglyceride concentration and gene expression were examined by biochemical analysis and QPCR. Results: Our results revealed that CD38 deficiency significantly elevated the intracellular glutathione (GSH) concentration and GSH/GSSG ratio, decreased the contents of free fatty acids and increased intracellular NAD⁺ level in heart from CD38^-/- mice fed with HFD. In addition, in vitro knockdown of CD38 significantly attenuated OA-induced cellular injury, ROS production and lipid synthesis. Furthermore, the expression of mitochondrial deacetylase Sirt3 as well as its target genes FOXO3 and SOD2 were markedly upregulated in the H9C2 cell lines after OA stimulation. In contrast, the expressions of NOX2 and NOX4 were significantly decreased in the cells after OA stimulation. Conclusion: Our results demonstrated that CD38 deficiency protected heart from HFD-induced oxidative stress via activating Sirt3/FOXO3-mediated anti-oxidative stress pathway.

Antitoxic effect of Veratrilla baillonii on the acute toxicity in mice induced by Aconitum brachypodum, one of the genus Aconitum

Ethnopharmacological relevanceAconitum brachypodum Diels (Family Ranunculaceae) is well known for both its good therapy and high toxicity in Yunnan and Sichuan provinces in China. Noticeably, Veratrilla baillonii Franch (Family Gentianaceae), an ethnodrug used by Naxi and Lisu nationalities in Yunnan Province, has been widely considered to possess antitoxic effects on Aconitum plants in herbal therapy and folklore medicines. Materials and methodsThe present study was conducted to determine the detoxic activities of the water decoction of Veratrilla baillonii Franch (WVBF) on the the chloroform fraction of Aconitum brachypodum Diels (CFA) induced acute toxicity in mice. The physiological (symptoms, body weight, etc.) as well as pathological and clinical biochemistry parameters were assessed and used as the markers for the toxicity. 1H nuclear magnetic resonance (NMR) based metabolic approach was adopted to further discuss the mechanism. ResultsThe acute poisoning effects of CFA on mice were observed at doses of 20–62.5mgkg−1, resulting in an oral median lethal dose (LD50) of 41.3mgkg−1. Histologically, distinct degenerative changes of the heart, liver and kidney were observed. The biochemistry parameters in the serum as well as metabolites in heart and brain were also altered. However, WVBF (25–200mg/kg) attenuated all the acute toxicity and pathological changes, properly regulated the biochemistry parameters, and reversed the concentration alterations for some metabolites in the heart and brain of mice induced by 40mg/kg of CFA to a certain extent. ConclusionsWVBF significantly reduced the onset of the CFA toxicity. This study may contribute to further understanding of the toxicological and pharmacological profiles of Aconitum brachypodum and the detoxic property of Veratrilla baillonii.

Regional Sympathetic Blockade Attenuates Activation of Intestinal Macrophages and Reduces Gut Barrier Failure

Endotoxin-induced activation of monocytes may lead to extravasation of cells, excessive production of nitric oxide, and subsequent epithelial injury in the gut. Regional sympathetic blockade by means of thoracic epidural anesthesia has been implicated to protect the epithelial barrier. This study tested the hypothesis that thoracic epidural anesthesia decreases epithelial permeability by attenuating monocytic production of nitric oxide and nitrosative stress. Rats were anesthetized, hemodynamically monitored, and mechanically ventilated. Endotoxemia was induced by an intravenous bolus injection of Escherichia coli lipopolysaccharide. Either lidocaine 2% or normal saline was injected as a bolus, followed by a continuous infusion via an epidural catheter. Three hundred minutes after injection of lipopolysaccharide or normal saline, gut epithelial permeability to fluorescein isothiocyanate-dextran (4 kDa), intestinal expression of inducible nitric oxide synthase by macrophages, and lipid peroxidation represented by 8-isoprostane tissue concentration were quantified. Thoracic epidural anesthesia significantly attenuated the endotoxin-induced increases in gut epithelial permeability (437 [293, 492] vs. 628 [532, 1,042] ng/ml, median [quartiles], P = 0.03), expression of nitric oxide synthase (2 [1,2] vs. 7 [5,8] cells per 384 µm(2), P = 0.003), macrophage infiltration, and lipid peroxidation (22,460 ± 11,476 vs. 37,840 ± 17,551 pg/ml, mean ± SD, P = 0.05). Thoracic epidural anesthesia attenuates endotoxin-induced gut epithelial injury. This is likely due to a decrease in monocytic extravasation and intestinal nitrosative stress. As possible mechanisms, direct nerve-immune interplay, a reduction in plasma catecholamines, or a systemic lidocaine effect has to be considered.

Cytochrome P450 enzymes and the heart

The cytochrome P450 monooxygenase system (CYP) is a multigene superfamily of heme-thiolate enzymes, which are important in the metabolism of foreign and endogenous compounds. Genetic variations, drug interactions, or pathophysiological factors can lead to reduced, absent, or increased enzymatic activity. This altered CYP activity greatly influences an individual's response to therapeutic treatment. What is not known is the impact of these changes on the many functional roles of CYP in physiological and pathophysiological processes of the heart. Many extrahepatic tissues, like heart, contain active P450 enzymes but lack information regarding their role in cellular injury or homeostasis. Much of our current knowledge about cardiac CYP has been limited to studies investigating the role of fatty acid metabolites in heart. Traditional risk factors including diabetes, smoking, and hypertension have well established links to cardiovascular disease. And new evidence strongly suggests exposure to chemicals and other environmental agents has a profound impact on the cardiovascular system. These risk factors can independently affect the expression and activity of CYP enzymes. Therefore, altered CYP activity is important from a detoxification as well as a bioactivation perspective. Considering CYP, interactions are greatly dependent on inherited differences or acquired changes in enzyme activity further research into their potential impact on pathogenesis, risk assessment, and therapy of heart disease is warranted. This review explores the expression of CYP isoforms, their functional roles, and the effects of genetic variation in the heart.

Presence of Specific 11C-meta-Hydroxyephedrine Retention in Heart, Lung, Pancreas, and Brown Adipose Tissue

(11)C-meta-Hydroxyephedrine (HED) is used in cardiac PET as an index of norepinephrine (NE) reuptake transporter (NET) density and synaptic NE levels. Whereas cardiac uptake is well documented, tracer retention in other tissues with rich noradrenergic innervation is unclear. Dysfunctional sympathetic nervous system (SNS) function in extracardiac metabolic storage tissues (i.e., adipose tissue and skeletal muscle) and endocrine organs contributes to several disorders. The aim of this study was to determine the potential of HED as an index of NE function in brown adipose tissue, lung, pancreas, skeletal muscle, and kidney by identifying NET-specific retention and determining the presence of radiolabeled metabolites. Male Sprague-Dawley rats were administered HED and sacrificed at 30 min after tracer injection. Tissues were rapidly excised and counted for radioactivity, and relative tracer retention was quantified. Pretreatment with NET inhibitors established specific HED accumulation. The effect of elevated NE was tested by subcutaneous minipump NE infusion or inhibition of monoamine oxidase. Column-switch high-performance liquid chromatography (HPLC) was used to analyze the presence of radiolabeled metabolites in heart, brown adipose tissue, pancreas, and plasma. NET-specific retention was observed in heart, brown adipose tissue, lung, and pancreas but not in liver, skeletal muscle, or kidney. A dose-dependent response of HED accumulation to treatments elevating NE levels was established in tissues exhibiting specific uptake. At 30 min after tracer administration, HPLC analysis revealed 93%-95% of total radioactivity signal derived from unchanged HED in heart, pancreas, and brown adipose tissue compared with 61% +/- 8% unchanged HED in plasma. In addition to the heart, lung, pancreas, and brown adipose tissue exhibit specific and NE-responsive uptake of HED, supporting the potential for novel PET studies of SNS integrity in these tissues.

Interstitial purine metabolites in hearts with LV remodeling.

The myocardial ATP concentration is significantly decreased in failing hearts, which may be related to the progressive loss of the myocardial total adenine nucleotide pool. The total myocardial interstitial purine metabolites (IPM) in the dialysate of interstitial fluid could reflect the tissue ATP depletion. In rats, postmyocardial infarction (MI) left ventricular (LV) remodeling was induced by ligation of the coronary artery. Cardiac microdialysis was employed to assess changes of IPM in response to graded beta-adrenergic stimulation with isoproterenol (Iso) in myocardium of hearts with post-MI LV remodeling (MI group) or hearts with sham operation (sham group). The dialysate samples were analyzed for adenosine, inosine, hypoxanthine, xanthine, and uric acid. LV volume was greater in the MI group (2.2 +/- 0.2 ml/kg) compared with the sham group (1.3 +/- 0.2 ml/kg, P < 0.05). Infarct size was 28 +/- 4%. The baseline dialysate level of uric acid was higher in the MI group (18.9 +/- 3.4 micromol) compared with the sham group (4.6 +/- 0.7 micromol, P < 0.01). During and after Iso infusion, the dialysate levels of adenosine, xanthine, and uric acid were all significantly higher in the MI group. Thus the level of IPM is increased in hearts with postinfarction LV remodeling both at baseline and during Iso infusion. These results suggest that the decreased myocardial ATP level in hearts with post-MI LV remodeling may be caused by the chronic depletion of the total adenine nucleotide pool.

Measurement of transmural distribution of phosphorus metabolites in the pig heart by 31P magnetic resonance spectroscopy.

We used the phase modulated rotating frame imaging technique to measure transmural distribution of phosphorus metabolites in 10 anaesthetised ventilated pigs using a double surface coil placed on the surface of the left ventricle. Anaesthesia was maintained in five animals with halothane, barbiturate and nitrous oxide and in five others with intravenous chloralose. 31Phosphorus spectra were acquired, gated to expiration and systole. From phantom experiments the resolution of the experiment was shown to be approximately 2 mm. The anatomical limits of the myocardium were identified by the appearance of 2,3-diphosphoglycerate peaks from red blood cells. The limits of the epicardium were confirmed by obtaining images after placing a phantom containing fluorophosphate on the surface of the heart. The endocardium was identified by inserting a small balloon catheter through the centre of the coil into the left ventricular cavity, filling it with 0.5 ml of fluorophosphate and pulling it gently against the endocardium. No transmural differences in phosphocreatine to ATP ratio were identified in the normal heart. The animals anaesthetised with chloralose showed a significantly higher phosphocreatine to ATP ratio compared to those anaesthetised with halothane and barbiturate. The chloralose animals tended to have a higher blood pressure and a lower heart rate when compared to the other animals. No transmural differences, however, were identified in either group. When regional ischaemia was produced using a snare to occlude the left coronary artery, phosphocreatine fell and the signal from the inorganic phosphate + 2,3-diphosphoglycerate region increased. The inner wall tended to become more acid compared to the outer wall during ischaemia. These experiments show that the phase modulated rotating frame imaging technique can be used to study the effects of changes in workload, ischaemia, or pharmacological intervention on transmural distribution of metabolites in the heart and thus help elucidate factors responsible for subendocardial vulnerability to stress.

Distribution of digoxin, digitoxin and their cardioactive metabolites in human heart and kidney tissue. A postmortem study

A method was developed for the specific determination of digoxin and digitoxin, as well as their semisynthetic derivatives and dependent cardioactive metabolites, in autopsy samples of heart and kidney. A collective of six patients on long-term treatment with therapeutic doses of beta-acetyldigoxin had a mean myocardial digoxin content of 46.1 +/- 25.0 ng/g (SD); kidney: 50.3 +/- 30.3 ng/g. Digoxigenin bisdigitoxoside represented the second most important metabolite in heart and kidney; digoxigenin monodigitoxoside and digoxigenin follow, respectively. In a collective of seven patients on maintenance treatment with digitoxin, the mean tissue levels were higher but the metabolic pattern was similar (myocardial digitoxin content: 78.9 +/- 38.4 ng/g, renal content: 104.1 +/- 44.1 ng/g). The amount of digoxin formed by hydroxylation under long-term treatment with digitoxin in heart and kidney were approximately 10 ng/g. A case of digoxin intoxication differed both in the tissue content and in the metabolic distribution.

Presence of α-Methyl-dopa metabolites in heart and brain of Guinea pigs treated with α-methyl-tyrosine

Recently it was shown that the rate-limiting step in noradrenaline (NA) biosynthesis is the hydroxylation of tyrosine to DOPA (1, 2). The enzyme which catalyzes this conversion, tyrosine hydroxylase, has been isolated from various tissues and studied extensively (3, 4, 5). Several tyrosine analogues and catechols were shown to inhibit tyrosine hydroxylase, and α-methyl-tyrosine (α-MT) can be considered as one of the most potent inhibitors (4, 6). α-MT lowers endogenous levels of NA and the mechanism of this depletion has been ascribed to a blockade of the enzymatic synthesis of NA at the tyrosine hydroxylase step (7). The present study shows that after giving α-MT the same α-methylated catecholamines are formed in vivo as after treatment with α-methyl-DOPA, but the concentration in heart and brain are lower.