Urinary FABP1 is a biomarker for impaired proximal tubular protein reabsorption and is synergistically enhanced by concurrent liver injury.

Urinary fatty acid binding protein 1 (FABP1, also known as liver-type FABP) has been implicated as a biomarker of acute kidney injury (AKI) in humans. However, the precise biological mechanisms underlying its elevation remain elusive. Here, we show that urinary FABP1 primarily reflects impaired protein reabsorption in proximal tubule epithelial cells (PTECs). Bilateral nephrectomy resulted in a marked increase in serum FABP1 levels, suggesting that the kidney is an essential organ for removing serum FABP1. Injected recombinant FABP1 was filtered through the glomeruli and robustly reabsorbed via the apical membrane of PTECs. Urinary FABP1 was significantly elevated in mice devoid of megalin, a giant endocytic receptor for protein reabsorption. Elevation of urinary FABP1 was also observed in patients with Dent disease, a rare genetic disease characterized by defective megalin function in PTECs. Urinary FABP1 levels were exponentially increased following acetaminophen overdose, with both nephrotoxicity and hepatotoxicity observed. FABP1-deficient mice with liver-specific overexpression of FABP1 showed a massive increase in urinary FABP1 levels upon acetaminophen injection, indicating that urinary FABP1 is liver-derived. Lastly, we employed transgenic mice expressing diphtheria toxin receptor (DT-R) either in a hepatocyte- or in a PTEC-specific manner, or both. Upon administration of diphtheria toxin (DT), massive excretion of urinary FABP1 was induced in mice with both kidney and liver injury, while mice with either injury type showed marginal excretion. Collectively, our data demonstrated that intact PTECs have a considerable capacity to reabsorb liver-derived FABP1 through a megalin-mediated mechanism. Thus, urinary FABP1, which is synergistically enhanced by concurrent liver injury, is a biomarker for impaired protein reabsorption in AKI. These findings address the use of urinary FABP1 as a biomarker of histologically injured PTECs that secrete FABP1 into primary urine, and suggest the use of this biomarker to simultaneously monitor impaired tubular reabsorption and liver function. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

FABP5 Is a Sensitive Marker for Lipid-Rich Macrophages in the Luminal Side of Atherosclerotic Lesions.

Lipid-rich macrophages in atherosclerotic lesions are thought to be derived from myeloid and vascular smooth muscle cells. A series of studies with genetic and pharmacological inhibition of fatty acid binding protein 4 (FABP4) and FABP5 and bone marrow transplant experiments with FABP4/5 deficient cells in mice have demonstrated that these play an important role in the development of atherosclerosis. However, it is still uncertain about the differential cell-type specificity and distribution between FABP4- and FABP5-expressing cells in early- and late-stage atherosclerotic lesions. In this study, we first explored spatial distribution of FABP4/5 in atherosclerotic lesions in apolipoprotein E deficient (ApoE-/-) mice. FABP4 was only marginally detected in early and advanced lesions, whereas FABP5 was abundantly expressed in these lesions. In advanced lesions, the FABP5-positive area was mostly restricted to the foam cell layer adjacent to the lumen above collagen and elastic fibers with a high signal/noise ratio. Oil red O (ORO) staining revealed that FABP5-positive cells were lipid-rich in early and advanced lesions. Together, most of lipid-rich FABP5-positive cells reside adjacent to the lumen above collagen and elastic fibers. We next studied involvement of FABP5 in lesion formation of atherosclerosis using ApoE-/- FABP5-/- mice. However, deletion of FABP5 did not affect the development of atherosclerosis. These findings, along with previous reports, suggest a novel notion that FABP5 is a sensitive marker for bone marrow-derived lipid-rich macrophages in the luminal side of atherosclerotic lesions, although its functional significance remains elusive.

Exercise endurance capacity is markedly reduced due to impaired energy homeostasis during prolonged fasting in FABP4/5 deficient mice.

BACKGROUND: Skeletal muscle prefers carbohydrate use to fatty acid (FA) use as exercise intensity increases. In contrast, skeletal muscle minimizes glucose use and relies more on FA during fasting. In mice deficient for FABP4 and FABP5 (double knockout (DKO) mice), FA utilization by red skeletal muscle and the heart is markedly reduced by the impairment of trans-endothelial FA transport, with an increase in glucose use to compensate for reduced FA uptake even during fasting. We attempted to determine whether prolonged fasting affects exercise performance in DKO mice, where constant glucose utilization occurs. RESULTS: A single bout of treadmill exercise was performed in the fed and fasted states. The initial speed was 10 m/min, and gradually increased by 5 m/min every 5 min up to 30 m/min until the mice stopped running. Running distance was significantly reduced by DKO genotype and prior fasting, leading to the shortest distance in fasted DKO mice. Levels of glycogen in skeletal muscle and the liver were nearly depleted in both WT and DKO mice during prolonged fasting prior to exercise. Levels of TG in skeletal muscle were not reduced by exercise in fasted DKO mice, suggesting that intramuscular TG was not utilized during exercise. Hypoglycaemia was accelerated in fasted DKO mice, and this acceleration could be due to constant glucose utilization by red skeletal muscle and the heart where FA uptake is diminished due to defective trans-endothelial FA transport. Taken together, energy supply from serum and storage in skeletal muscle were very low in fasted DKO mice, which could lead to a significant reduction in exercise performance. CONCLUSIONS: FABP4/5 have crucial roles in nutrient homeostasis during prolonged fasting for maintaining exercise endurance capacity.

Activation of pyruvate dehydrogenase by dichloroacetate has the potential to induce epigenetic remodeling in the heart.

Dichloroacetate (DCA) promotes pyruvate entry into the Krebs cycle by inhibiting pyruvate dehydrogenase (PDH) kinase and thereby maintaining PDH in the active dephosphorylated state. DCA has recently gained attention as a potential metabolic-targeting therapy for heart failure but the molecular basis of the therapeutic effect of DCA in the heart remains a mystery. Once-daily oral administration of DCA alleviates pressure overload-induced left ventricular remodeling. We examined changes in the metabolic fate of pyruvate carbon (derived from glucose) entering the Krebs cycle by metabolic interventions of DCA. (13)C6-glucose pathway tracing analysis revealed that instead of being completely oxidized in the mitochondria for ATP production, DCA-mediated PDH dephosphorylation results in an increased acetyl-CoA pool both in control and pressure-overloaded hearts. DCA induces hyperacetylation of histone H3K9 and H4 in a dose-dependent manner in parallel to the dephosphorylation of PDH in cultured cardiomyocytes. DCA administration increases histone H3K9 acetylation in in vivo mouse heart. Interestingly, DCA-dependent histone acetylation was associated with an up-regulation of 2.3% of genes (545 out of 23,474 examined). Gene ontology analysis revealed that these genes are highly enriched in transcription-related categories. This evidence suggests that sustained activation of PDH by DCA results in an overproduction of acetyl-CoA, which exceeds oxidation in the Krebs cycle and results in histone acetylation. We propose that DCA-mediated PDH activation has the potential to induce epigenetic remodeling in the heart, which, at least in part, forms the molecular basis for the therapeutic effect of DCA in the heart.

CD36 is indispensable for thermogenesis under conditions of fasting and cold stress.

Hypothermia can occur during fasting when thermoregulatory mechanisms, involving fatty acid (FA) utilization, are disturbed. CD36/FA translocase is a membrane protein which facilitates membrane transport of long-chain FA in the FA consuming heart, skeletal muscle (SkM) and adipose tissues. It also accelerates uptake of triglyceride-rich lipoprotein by brown adipose tissue (BAT) in a cold environment. In mice deficient for CD36 (CD36(-/-) mice), FA uptake is markedly reduced with a compensatory increase in glucose uptake in the heart and SkM, resulting in lower levels of blood glucose especially during fasting. However, the role of CD36 in thermogenic activity during fasting remains to be determined. In fasted CD36(-/-) mice, body temperature drastically decreased shortly after cold exposure. The hypothermia was accompanied by a marked reduction in blood glucose and in stores of triacylglycerols in BAT and of glycogen in glycolytic SkM. Biodistribution analysis using the FA analogue (125)I-BMIPP and the glucose analogue (18)F-FDG revealed that uptake of FA and glucose was severely impaired in BAT and glycolytic SkM in cold-exposed CD36(-/-) mice. Further, induction of the genes of thermogenesis in BAT was blunted in fasted CD36(-/-) mice after cold exposure. These findings strongly suggest that CD36(-/-) mice exhibit pronounced hypothermia after fasting due to depletion of energy storage in BAT and glycolytic SkM and to reduced supply of energy substrates to these tissues. Our study underscores the importance of CD36 for nutrient homeostasis to survive potentially life-threatening challenges, such as cold and starvation.

Capillary endothelial fatty acid binding proteins 4 and 5 play a critical role in fatty acid uptake in heart and skeletal muscle.

OBJECTIVE: Fatty acids (FAs) are the major substrate for energy production in the heart. Here, we hypothesize that capillary endothelial fatty acid binding protein 4 (FABP4) and FABP5 play an important role in providing sufficient FAs to the myocardium. APPROACH AND RESULTS: Both FABP4/5 were abundantly expressed in capillary endothelium in the heart and skeletal muscle. The uptake of a FA analogue, 125I-15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid, was significantly reduced in these tissues in double-knockout (DKO) mice for FABP4/5 compared with wild-type mice. In contrast, the uptake of a glucose analogue, 18F-fluorodeoxyglucose, was remarkably increased in DKO mice. The expression of transcripts for the oxidative catabolism of FAs was reduced during fasting, whereas transcripts for the glycolytic pathway were not altered in DKO hearts. Notably, metabolome analysis revealed that phosphocreatine and ADP levels were significantly lower in DKO hearts, whereas ATP content was kept at a normal level. The protein expression levels of the glucose transporter Glut4 and the phosphorylated form of phosphofructokinase-2 were increased in DKO hearts, whereas the phosphorylation of insulin receptor-ß and Akt was comparable between wild-type and DKO hearts during fasting, suggesting that a dramatic increase in glucose usage during fasting is insulin independent and is at least partly attributed to the post-transcriptional and allosteric regulation of key proteins that regulate glucose uptake and glycolysis. CONCLUSIONS: Capillary endothelial FABP4/5 are required for FA transport into FA-consuming tissues that include the heart. These findings identify FABP4/5 as promising targets for controlling the metabolism of energy substrates in FA-consuming organs that have muscle-type continuous capillary.

Empagliflozin induces the transcriptional program for nutrient homeostasis in skeletal muscle in normal mice.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) improve heart failure (HF) outcomes across a range of patient characteristics. A hypothesis that SGLT2i induce metabolic change similar to fasting has recently been proposed to explain their profound clinical benefits. However, it remains unclear whether SGLT2i primarily induce this change in physiological settings. Here, we demonstrate that empagliflozin administration under ad libitum feeding did not cause weight loss but did increase transcripts of the key nutrient sensors, AMP-activated protein kinase and nicotinamide phosphoribosyltransferase, and the master regulator of mitochondrial gene expression, PGC-1α, in quadriceps muscle in healthy mice. Expression of these genes correlated with that of PPARα and PPARδ target genes related to mitochondrial metabolism and oxidative stress response, and also correlated with serum ketone body ß-hydroxybutyrate. These results were not observed in the heart. Collectively, this study revealed that empagliflozin activates transcriptional programs critical for sensing and adaptation to nutrient availability intrinsic to skeletal muscle rather than the heart even in normocaloric condition. As activation of PGC-1α is sufficient for metabolic switch from fatigable, glycolytic metabolism toward fatigue-resistant, oxidative mechanism in skeletal muscle myofibers, our findings may partly explain the improvement of exercise tolerance in patients with HF receiving empagliflozin.

Pathophysiologic Contributions of Visceral Adiposity to Left Ventricular Diastolic Dysfunction.

BACKGROUND: Visceral fat produces inflammatory cytokines and may play a major role in heart failure with preserved ejection fraction (HFpEF). However, little data exist regarding how qualitative and quantitative abnormalities of visceral fat would contribute to left ventricular diastolic dysfunction (LVDD). METHODS: We studied 77 participants who underwent open abdominal surgery for intra-abdominal tumors (LVDD, n = 44; controls without LVDD, n = 33). Visceral fat samples were obtained during the surgery, and mRNA levels of inflammatory cytokines were measured. Visceral and subcutaneous fat areas were measured using abdominal computed tomography. RESULTS: Patients with significant LVDD had greater LV remodeling and worse LVDD than controls. While body weight, body mass index, and subcutaneous fat area were similar in patients with LVDD and controls, the visceral fat area was larger in patients with LVDD than in controls. The visceral fat area was correlated with BNP levels, LV mass index, mitral e' velocity, and E/e' ratio. There were no significant differences in the mRNA expressions of visceral adipose tissue cytokines (IL-2, -6, -8, and -1ß, TNFα, CRP, TGFß, IFNγ, leptin, and adiponectin) between the groups. CONCLUSIONS: Our data may suggest the pathophysiological contribution of visceral adiposity to LVDD.

Notch signaling pathway enhances bone morphogenetic protein 2 (BMP2) responsiveness of Msx2 gene to induce osteogenic differentiation and mineralization of vascular smooth muscle cells.

Vascular calcification is regulated in a process similar to bone formation. BMP2 (bone morphogenetic protein 2) is essential for osteoblastic differentiation of mesenchymal progenitor cells and thus has been implicated in the development of vascular calcification. Here we examined whether Notch signaling interacts with BMP2 signaling to regulate osteogenic differentiation and mineralization of vascular smooth muscle cells (SMCs). BMP2 alone scarcely induced the expression of alkaline phosphatase (ALP), an ectoenzyme crucially required for active biomineralization, in human aortic SMCs (HASMCs), despite its strong induction in osteoblast precursor MC3T3-E1 cells. Notably, overexpression of the Notch1 intracellular domain (N1-ICD) markedly enhanced BMP2-mediated induction of ALP activity and mineralization of HASMCs. In HASMCs, expression of Msx2 gene, a well documented BMP2 target gene in osteoblasts, was barely induced by BMP2 alone, and N1-ICD clearly enhanced the BMP2-driven Msx2 gene expression. Deletion and site-directed mutation analysis of Msx2 gene promoter revealed that the RBPJk-binding site was necessary for BMP2 responsiveness. Using the RBPJk-deficient cells and siRNA for RBPJk, we showed that RBPJk was required for BMP2 induction of Msx2 gene expression and ALP activity. Moreover, we showed that Smad1, a transcription factor downstream of BMP2 signaling, interacted with N1-ICD to form a complex within the Msx2 promoter. Immunohistochemistry of human calcifying atherosclerotic plaques revealed colocalized expression of Notch1, BMP2, and Msx2. These results indicate that the Notch intracellular domain·RBPJk complex enhances the BMP2-induced Msx2 gene expression by cooperating with Smad1 and suggest that Notch signaling makes vascular SMC responsive to BMP2 and promotes vascular calcification.

Compound and digenic heterozygosity in desmosome genes as a cause of arrhythmogenic right ventricular cardiomyopathy in Japanese patients.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary disorder mostly caused by desmosome gene mutations. Recent comprehensive desmosome mutation analyses of Caucasian ARVC patients have revealed the presence of not only a single heterozygous mutation, but also compound and digenic heterozygosity. However, the genetic basis of Japanese ARVC remains poorly elucidated. METHODS AND RESULTS: The subjects were 7 definite and 1 possible ARVC probands (6 males, 16-76 years of age), and their family members. Genetic screening for major ARVC-causing genes (junction plakoglobin, desmoplakin, plakophilin-2 (PKP2), desmoglein-2 (DSG2), and desmocollin-2) was performed. We identified 3 cases of compound heterozygosities (Case 1: DSG2 S194L and DSG2 R292C; Case 2: PKP2 2489+1G>A and PKP2 D812N; Case 3: PKP2 M565R and PKP2 D812N) and 1 of digenic heterozygosity (Case 4: PKP2 1728_1729insGATG and DSG2 R292C) among the definite ARVC patients. All family members we investigated have remained asymptomatic. They carried, if any, only a single variant, indicating that the probands carry in trans compound heterozygosity. These results suggest that each of these variants alone may not be sufficient and second variants may be required to manifest overt ARVC in Japanese patients. CONCLUSIONS: Our comprehensive genetic analysis of desmosome genes identified 3 cases of compound heterozygosities in trans and 1 of digenic heterozygosity among 7 definite Japanese ARVC patients, providing novel insights into the genetic basis of Japanese ARVC.

Notch mediates TGF alpha-induced changes in epithelial differentiation during pancreatic tumorigenesis.

Notch signaling regulates cell fate decisions in a wide variety of adult and embryonic tissues. Here we show that Notch pathway components and Notch target genes are upregulated in invasive pancreatic cancer, as well as in pancreatic cancer precursors from both mouse and human. In mouse pancreas, ectopic Notch activation results in accumulation of nestin-positive precursor cells and expansion of metaplastic ductal epithelium, previously identified as a precursor lesion for pancreatic cancer. Notch is also activated as a direct consequence of EGF receptor activation in exocrine pancreas and is required for TGF alpha-induced changes in epithelial differentiation. These findings suggest that Notch mediates the tumor-initiating effects of TG alpha by expanding a population of undifferentiated precursor cells.

Azelnidipine inhibits Msx2-dependent osteogenic differentiation and matrix mineralization of vascular smooth muscle cells.

Vascular calcification is an active and regulated process that is similar to bone formation. While calcium channel blockers (CCBs) have been shown to improve outcomes in atherosclerotic vascular disease, it remains unknown whether CCBs have an effect on the process of vascular calcification. Here we investigated whether CCBs inhibit osteogenic differentiation and matrix mineralization of vascular smooth muscle cells induced by Msx2, a key factor of vascular calcification. Human aortic smooth muscle cells (HASMCs) were transduced with adenovirus expressing MSX2 and were treated with 3 distinct CCBs. Azelnidipine, a dihydropyridine subclass of CCBs, significantly decreased alkaline phosphatase (ALP) activity of Msx2-overexpressed HASMCs, whereas verapamil and diltiazem had no effect. Furthermore, azelnidipine, but not verapamil and diltiazem, significantly decreased matrix mineralization of Msx2-overexpressing HASMCs. Azelnidipine significantly attenuated the induction of ALP gene expression by Msx2, a key transcription factor in osteogenesis, while it did not reduce enzymatic activity of ALP. Furthermore, azelnidipine inhibited the ability of Msx2 to activate the ALP gene, but had no effect on Notch-induced Msx2 expression. Given that L-type calcium channels are equally blocked by these CCBs, our results suggest that azelnidipine inhibits the Msx2-dependent process of vascular calcification by mechanisms other than inhibition of calcium channel activity.

Ketone body and FGF21 coordinately regulate fasting-induced oxidative stress response in the heart.

Ketone body ß-hydroxybutyrate (ßOHB) and fibroblast growth factor-21 (FGF21) have been proposed to mediate systemic metabolic response to fasting. However, it remains elusive about the signaling elicited by ketone and FGF21 in the heart. Stimulation of neonatal rat cardiomyocytes with ßOHB and FGF21 induced peroxisome proliferator-activated receptor α (PPARα) and PGC1α expression along with the phosphorylation of LKB1 and AMPK. ßOHB and FGF21 induced transcription of peroxisome proliferator-activated receptor response element (PPRE)-containing genes through an activation of PPARα. Additionally, ßOHB and FGF21 induced the expression of Nrf2, a master regulator for oxidative stress response, and catalase and Ucp2 genes. We evaluated the oxidative stress response gene expression after 24 h fast in global Fgf21-null (Fgf21-/-) mice, cardiomyocyte-specific FGF21-null (cmFgf21-/-) mice, wild-type (WT), and Fgf21fl/fl littermates. Fgf21-/- mice but not cmFgf21-/- mice had unexpectedly higher serum ßOHB levels, and higher expression levels of PPARα and oxidative stress response genes than WT mice or Fgf21fl/fl littermates. Notably, expression levels of oxidative stress response genes were significantly correlated with serum ßOHB and PGC1α levels in both WT and Fgf21-/- mice. These findings suggest that fasting-induced ßOHB and circulating FGF21 coordinately regulate oxidative stress response gene expression in the heart.

Notch induces myofibroblast differentiation of alveolar epithelial cells via transforming growth factor-{beta}-Smad3 pathway.

Notch is an ancient cell-signaling system that regulates the specification of cell fate. This study examined the role of Notch in the epithelial-mesenchymal transition (EMT) and myofibroblast differentiation of cultured RLE-6TN cells (i.e., rat alveolar epithelial cells). The activation of Notch, either by ectopic expression of the Notch intracellular domain or by the co-culture of RLE-6TN cells with L-Jagged1 cells, induces the expression of smooth muscle α-actin (SMA) and other mesenchymal marker genes (collagen I and vimentin), and reduces the expression of epithelial marker genes (E-cadherin, occludin, and zonula occludens-1). The pharmacologic inhibition of the endogenous Notch signal significantly inhibited the transforming growth factor-ß (TGF-ß)-induced expression of SMA. Cell migratory capacity was increased by Notch. Luciferase assays revealed that the CC(A/T)(6)GG (CArG) box and the TGF-ß control element (TCE) are required for Notch-induced SMA gene transcription. DNA microarray analysis revealed that members of the TGF-ß family as well as Jagged1 were induced in RLE-6TN cells by Notch. Western blot analysis showed that Notch induced the phosphorylation of Smad3, and the TGF-ß receptor type I/activin receptor-like kinase 5 (ALK5) kinase inhibitor SB431542 markedly reduced the Notch-induced expression of SMA. Enzyme-linked immunosorbent assays confirmed the production of TGF-ß1 from RLE-6TN cells by Notch. Immunohistochemistry of a bleomycin-induced model of pulmonary fibrosis and lung specimens from patients with idiopathic interstitial pneumonias showed that Notch was strongly expressed in myofibroblasts, identified as SMA-positive cells. These data indicate that Notch induces myofibroblast differentiation through a TGF-ß-Smad3 pathway that activates SMA gene transcription in a CArG-dependent and TCE-dependent manner in alveolar epithelial cells. Our data also imply that Notch induces the EMT phenotype, with increased migratory behavior in pulmonary fibrosis.

Hypoxia-inducible factor-1α mediates TGF-ß-induced PAI-1 production in alveolar macrophages in pulmonary fibrosis.

Hypoxia-inducible factor-1α (HIF-1α), a transcription factor that functions as a master regulator of oxygen homeostasis, has been implicated in fibrinogenesis. Here, we explore the role of HIF-1α in transforming growth factor-ß (TGF-ß) signaling by examining the effects of TGF-ß(1) on the expression of plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of lung tissue from a mouse bleomycin (BLM)-induced pulmonary fibrosis model revealed that expression of HIF-1α and PAI-1 was predominantly induced in alveolar macrophages. Real-time RT-PCR and ELISA analysis showed that PAI-1 mRNA and activated PAI-1 protein level were strongly induced 7 days after BLM instillation. Stimulation of cultured mouse alveolar macrophages (MH-S cells) with TGF-ß(1) induced PAI-1 production, which was associated with HIF-1α protein accumulation. This accumulation of HIF-1α protein was inhibited by SB431542 (type I TGF-ß receptor/ALK receptor inhibitor) but not by PD98059 (MEK1 inhibitor) and SB203580 (p38 MAP kinase inhibitor). Expression of prolyl-hydroxylase domain (PHD)-2, which is essential for HIF-1α degradation, was inhibited by TGF-ß(1), and this decrease was abolished by SB431542. TGF-ß(1) induction of PAI-1 mRNA and its protein expression were significantly attenuated by HIF-1α silencing. Transcriptome analysis by cDNA microarray of MH-S cells after HIF-1α silencing uncovered several pro-fibrotic genes whose regulation by TGF-ß(1) required HIF-1α, including platelet-derived growth factor-A. Taken together, these findings expand our concept of the role of HIF-1α in pulmonary fibrosis in mediating the effects of TGF-ß(1) on the expression of the pro-fibrotic genes in activated alveolar macrophages.

Identification of six novel SCN5A mutations in Japanese patients with Brugada syndrome.

Mutations in SCN5A are linked to Brugada syndrome in approximately 20% of all cases (BrS1). Several dozen distinct SCN5A mutations in BrS1 have been associated with the increased risk of cardiac arrhythmias. However, the genotype-phenotype relationship remains elusive. The current study analyzed the SCN5A gene to elucidate the potential variability of clinical features in Japanese BrS1 subjects. Subjects of the present study included 30 probands (25 male subjects, 45 ± 15 years of age) with Brugada-pattern ECG. Seven patients had been resuscitated from cardiopulmonary arrest (CPA group). Another 10 patients had a history of syncope (Sy group), and 13 more remain asymptomatic (Asy group). We identified 8 different SCN5A mutations, including 6 novel mutations (CPA group: 1/7, Sy group: 3/10, Asy group: 4/13). An A735E mutation (located at segment (S)1 in domain (D)2) was identified in the CPA group. A novel splice acceptor site mutation (c.393-1c>t), which may produce a prematurely truncated protein, was identified in the Sy group. An E1784K mutation (C-terminus) and a novel mutation V1951M (C-terminus) were also identified in the Sy group. Four novel missense mutations, A586T (D1-D2 linker), R689H (D1-D2 linker), S1553R (S1-S2 in D4), and Q1706H (S5-Pore in D4) were identified in the Asy group. These data may help us understand the genetic heterogeneity of BrS1, which is more prevalent in Japanese than in whites and other ethnic groups.

Cardiac Metabolism and Contractile Function in Mice with Reduced Trans-Endothelial Fatty Acid Transport.

The heart is a metabolic omnivore that combusts a considerable amount of energy substrates, mainly long-chain fatty acids (FAs) and others such as glucose, lactate, ketone bodies, and amino acids. There is emerging evidence that muscle-type continuous capillaries comprise the rate-limiting barrier that regulates FA uptake into cardiomyocytes. The transport of FAs across the capillary endothelium is composed of three major steps-the lipolysis of triglyceride on the luminal side of the endothelium, FA uptake by the plasma membrane, and intracellular FA transport by cytosolic proteins. In the heart, impaired trans-endothelial FA (TEFA) transport causes reduced FA uptake, with a compensatory increase in glucose use. In most cases, mice with reduced FA uptake exhibit preserved cardiac function under unstressed conditions. When the workload is increased, however, the total energy supply relative to its demand (estimated with pool size in the tricarboxylic acid (TCA) cycle) is significantly diminished, resulting in contractile dysfunction. The supplementation of alternative fuels, such as medium-chain FAs and ketone bodies, at least partially restores contractile dysfunction, indicating that energy insufficiency due to reduced FA supply is the predominant cause of cardiac dysfunction. Based on recent in vivo findings, this review provides the following information related to TEFA transport: (1) the mechanisms of FA uptake by the heart, including TEFA transport; (2) the molecular mechanisms underlying the induction of genes associated with TEFA transport; (3) in vivo cardiac metabolism and contractile function in mice with reduced TEFA transport under unstressed conditions; and (4) in vivo contractile dysfunction in mice with reduced TEFA transport under diseased conditions, including an increased afterload and streptozotocin-induced diabetes.

Pathophysiological and diagnostic importance of fatty acid-binding protein 1 in heart failure with preserved ejection fraction.

Elevated intracardiac pressure at rest and/or exercise is a fundamental abnormality in heart failure with preserved ejection fraction (HFpEF). Fatty acid-binding protein 1 (FABP1) is proposed to be a sensitive biomarker for liver injury. We sought to determine whether FABP1 at rest would be elevated in HFpEF and would correlate with echocardiographic markers of intracardiac pressures at rest and during exercise. In this prospective study, subjects with HFpEF (n = 22) and control subjects without HF (n = 23) underwent resting FABP1 measurements and supine bicycle exercise echocardiography. Although levels of conventional hepatic enzymes were similar between groups, FABP1 levels were elevated in HFpEF compared to controls (45 [25-68] vs. 18 [14-24] ng/mL, p = 0.0008). FABP1 levels were correlated with radiographic and blood-based markers of congestion, hemodynamic derangements during peak exercise (E/e', r = 0.50; right atrial pressure, r = 0.35; pulmonary artery systolic pressure, r = 0.46), reduced exercise cardiac output (r = - 0.49), and poor exercise workload achieved (r = - 0.40, all p < 0.05). FABP1 distinguished HFpEF from controls with an area under the curve of 0.79 (p = 0.003) and had an incremental diagnostic value over the H2FPEF score (p = 0.007). In conclusion, FABP1 could be a novel hepatic biomarker that associates with hemodynamic derangements, reduced cardiac output, and poor exercise capacity in HFpEF.

Prognostic impact of elevated fatty acid-binding protein 1 in patients with heart failure.

AIMS: Few biomarkers to evaluate pathophysiological changes in extra-cardiac tissues have been identified in patients with heart failure (HF). Fatty acid-binding protein 1 (FABP), also known as liver FABP, is predominantly expressed in the liver. Circulating FABP1 has been proposed to be a sensitive biomarker for liver injury. However, little is known about the potential role of FABP1 as a biomarker for HF. METHODS AND RESULTS: Measurements of serum FABP1 and echocardiography were performed in subjects with compensated HF (n = 162) and control subjects without HF (n = 20). Patients were prospectively followed-up for a composite outcome of all-cause mortality or HF hospitalization. Compared with control subjects, levels of FABP1 were elevated in HF patients [7.9 (6.4-11.7) vs. 17.6 (10.4-28.9) ng/mL, P < 0.0001]. There were significant correlations between FABP1 levels and estimated right ventricular systolic pressure and right atrial pressure. During a median follow-up of 12.0 months, there were 55 primary composite endpoints in the HF cohort. The highest FABP1 tertile was associated with a three-fold increased risk of the composite outcome compared with the lowest tertile [95% confidence interval (1.46-6.68), P = 0.003], but other conventional hepatobiliary markers did not predict the outcome. After adjusting for age, sex, atrial fibrillation, and N-terminal pro-B-type natriuretic peptide levels, serum FABP1 remained independently associated with the outcome. Adding FABP1 to the model based on clinical factors and N-terminal pro-B-type natriuretic peptide significantly improved the prognostic value (global χ2 20.8 vs. 15.5, P = 0.01). CONCLUSION: Serum FABP1 levels are elevated in compensated HF patients, and the magnitude of elevation is independently associated with pulmonary hypertension, right atrial hypertension, and worse clinical outcomes. FABP1 may serve as a new potential biomarker for the assessment of hitherto unrecognized derangement of cardio-hepatic interaction in HF.

Reduced Fatty Acid Use from CD36 Deficiency Deteriorates Streptozotocin-Induced Diabetic Cardiomyopathy in Mice.

Cardiac dysfunction is induced by multifactorial mechanisms in diabetes. Deranged fatty acid (FA) utilization, known as lipotoxicity, has long been postulated as one of the upstream events in the development of diabetic cardiomyopathy. CD36, a transmembrane glycoprotein, plays a major role in FA uptake in the heart. CD36 knockout (CD36KO) hearts exhibit reduced rates of FA transport with marked enhancement of glucose use. In this study, we explore whether reduced FA use by CD36 ablation suppresses the development of streptozotocin (STZ)-induced diabetic cardiomyopathy. We found that cardiac contractile dysfunction had deteriorated 16 weeks after STZ treatment in CD36KO mice. Although accelerated glucose uptake was not reduced in CD36KO-STZ hearts, the total energy supply, estimated by the pool size in the TCA cycle, was significantly reduced. The isotopomer analysis with 13C6-glucose revealed that accelerated glycolysis, estimated by enrichment of 13C2-citrate and 13C2-malate, was markedly suppressed in CD36KO-STZ hearts. Levels of ceramides, which are cardiotoxic lipids, were not elevated in CD36KO-STZ hearts compared to wild-type-STZ ones. Furthermore, increased energy demand by transverse aortic constriction resulted in synergistic exacerbation of contractile dysfunction in CD36KO-STZ mice. These findings suggest that CD36KO-STZ hearts are energetically compromised by reduced FA use and suppressed glycolysis; therefore, the limitation of FA utilization is detrimental to cardiac energetics in this model of diabetic cardiomyopathy.