Troponin T amino acid mutation (ΔK210) knock-in mice as a neonatal dilated cardiomyopathy model.

BACKGROUND: Dilated cardiomyopathy (DCM) in children is often associated with poor morbidity and mortality and exhibits distinct pathological entities from those of adult DCM. Owing to the limited number of patients and the lack of a good animal model, the molecular mechanisms underlying pediatric DCM remain poorly understood. The purpose of this study is to establish an animal model of neonatal DCM and identify early progression factors. METHODS: Cardiac phenotypes and comprehensive gene expression profiles in homozygous ΔK210 knock-in (TNNT2ΔK210/ΔK210) mice were analyzed and compared to TNNT2+/ΔK210 and wild-type mice at 0 days and 1 week of age. RESULTS: Immediately after birth, the cardiac weight in TNNT2ΔK210/ΔK210 mice was already increased compared to that in TNNT2+/ΔK210 and wild-type mice. Echocardiographic examination of 0-day-old and 1-week-old TNNT2ΔK210/ΔK210 mice revealed similar phenotypes of pediatric DCM. In addition, several genes were significantly upregulated in the ventricular tissues of TNNT2ΔK210/ΔK210 mice, and the KEGG PATHWAY analysis revealed several important pathways such as cancer and focal adhesion that might be associated with the pathogenesis and development of DCM. CONCLUSIONS: TNNT2ΔK210/ΔK210 mice have already developed DCM at birth, indicating that they should be an excellent animal model to identify early progression factors of DCM. IMPACT: TNNT2ΔK210/ΔK210 mice are excellent animal model for DCM. TNNT2ΔK210/ΔK210 mice are excellent animal model to identify early progression factors of DCM. KEGG PATHWAY analysis revealed that several important pathways such as cancer and focal adhesion might be associated with the pathogenesis and development of neonatal DCM.

Evaluation of Ductal Tissue in Coarctation of the Aorta Using X-Ray Phase-Contrast Tomography.

We assessed the histological accuracy of X-ray phase-contrast tomography (XPCT) and investigated three-dimensional (3D) ductal tissue distribution in coarctation of the aorta (CoA) specimens. We used nine CoA samples, including the aortic isthmus, ductus arteriosus (DA), and their confluences. 3D images were obtained using XPCT. After scanning, the samples were histologically evaluated using elastica van Gieson (EVG) staining and transcription factor AP-2 beta (TFAP2B) immunostaining. XPCT sectional images clearly depicted ductal tissue distribution as low-density areas. In comparison with EVG staining, the mass density of the aortic wall positively correlated with elastic fiber formation (R = 0.69, P < 0.001). TFAP2B expression was consistent with low-density area including intimal thickness on XPCT images. On 3D imaging, the distances from the DA insertion to the distal terminal of the ductal media and to the intima on the ductal side were 1.63 ± 0.22 mm and 2.70 ± 0.55 mm, respectively. In the short-axis view, the posterior extension of the ductal tissue into the aortic lumen was 79 ± 18% of the diameter of the descending aorta. In three specimens, the aortic wall was entirely occupied by ductal tissue. The ductal intima spread more distally and laterally than the ductal media. The contrast resolution of XPCT images was comparable to that of histological assessment. Based on the 3D images, we conclude that complete resection of intimal thickness, including the opposite side of the DA insertion, is required to eliminate residual ductal tissue and to prevent postoperative re-coarctation.

Pathophysiologic Role of Molecules Determining Arteriovenous Differentiation in Adult Life.

The structural differences between arteries and veins are genetically predetermined. Vascular identity markers, the molecular markers specific to veins and arteries, determine the differential development of vessels during embryogenesis and their expression persists in adult vessels. It is revealed that they can be reactivated under various pathophysiologic conditions even after vessel differentiation. Thus, once considered as quiescent in adults, vascular identity markers may actually play significant roles in vascular remodeling. Manipulation of vascular identity and the underlying molecular mechanisms might be a novel strategy to improve vascular remodeling for clinical application.

The serine/threonine-protein kinase/endoribonuclease IRE1α protects the heart against pressure overload-induced heart failure.

Heart failure is associated with induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The serine/threonine protein kinase/endoribonuclease IRE1α is a key protein in ER stress signal transduction. IRE1α activity can induce both protective UPR and apoptotic downstream signaling events, but the specific role for IRE1α activity in the heart is unknown. A major aim of this study was to characterize the specific contribution of IRE1α in cardiac physiology and pathogenesis. We used both cultured myocytes and a transgenic mouse line with inducible and cardiomyocyte-specific IRE1α overexpression as experimental models to achieve targeted IRE1α activation. IRE1α expression induced a potent but transient ER stress response in cardiomyocytes and did not cause significant effects in the intact heart under normal physiological conditions. Furthermore, the IRE1α-activated transgenic heart responding to pressure overload exhibited preserved function and reduced fibrotic area, associated with increased adaptive UPR signaling and with blunted inflammatory and pathological gene expression. Therefore, we conclude that IRE1α induces transient ER stress signaling and confers a protective effect against pressure overload-induced pathological remodeling in the heart. To our knowledge, this report provides first direct evidence of a specific and protective role for IRE1α in the heart and reveals an interaction between ER stress signaling and inflammatory regulation in the pathologically stressed heart.

Pyruvate dehydrogenase activation precedes the down-regulation of fatty acid oxidation in monocrotaline-induced myocardial toxicity in mice.

Fatty acid (FA) oxidation is impaired and glycolysis is promoted in the damaged heart. However, the factor(s) in the early stages of myocardial metabolic impairment remain(s) unclear. C57B6 mice were subcutaneously administered monocrotaline (MCT) in doses of 0.3 mg/g body weight twice a week for 3 or 6 weeks. Right and left ventricles at 3 and 6 weeks after administration were subjected to capillary electrophoresis-mass spectrometry metabolomic analysis. We also examined mRNA and protein levels of key metabolic molecules. Although no evidence of PH and right ventricular failure was found in the MCT-administered mice by echocardiographic and histological analyzes, the expression levels of stress markers such as TNFα and IL-6 were increased in right and left ventricles even at 3 weeks, suggesting that there was myocardial damage. Metabolites in the tricarboxylic acid (TCA) cycle were decreased and those in glycolysis were increased at 6 weeks. The expression levels of FA oxidation-related factors were decreased at 6 weeks. The phosphorylation level of pyruvate dehydrogenase (PDH) was significantly decreased at 3 weeks. FA oxidation and the TCA cycle were down-regulated, whereas glycolysis was partially up-regulated by MCT-induced myocardial damage. PDH activation preceded these alterations, suggesting that PDH activation is one of the earliest events to compensate for a subtle metabolic impairment from myocardial damage.

Cardiac myocyte p38α kinase regulates angiogenesis via myocyte-endothelial cell cross-talk during stress-induced remodeling in the heart.

Stress-induced p38 mitogen-activated protein kinase (MAPK) activity is implicated in pathological remodeling in the heart. For example, constitutive p38 MAPK activation in cardiomyocytes induces pathological features, including myocyte hypertrophy, apoptosis, contractile dysfunction, and fetal gene expression. However, the physiological function of cardiomyocyte p38 MAPK activity in beneficial compensatory vascular remodeling is unclear. This report investigated the functional role and the underlying mechanisms of cardiomyocyte p38 MAPK activity in cardiac remodeling induced by chronic stress. Using both in vitro and in vivo model systems, we found that p38 MAPK activity is required for hypoxia-induced pro-angiogenic activity from cardiomyocytes and that p38 MAPK activation in cardiomyocyte is sufficient to promote paracrine signaling-mediated, pro-angiogenic activity. We further demonstrate that VEGF is a paracrine factor responsible for the p38 MAPK-mediated pro-angiogenic activity from cardiomyocytes and that p38 MAPK pathway activation is sufficient for inducing VEGF secretion from cardiomyocytes in an Sp1-dependent manner. More significantly, cardiomyocyte-specific inactivation of p38α in mouse heart impaired compensatory angiogenesis after pressure overload and promoted early onset of heart failure. In summary, p38αMAPK has a critical role in the cross-talk between cardiomyocytes and vasculature by regulating stress-induced VEGF expression and secretion in cardiomyocytes. We conclude that as part of a stress-induced signaling pathway, p38 MAPK activity significantly contributes to both pathological and compensatory remodeling in the heart.

CCN3 secreted by prostaglandin E2 inhibits intimal cushion formation in the rat ductus arteriosus.

The ductus arteriosus (DA), an essential fetal shunt between the pulmonary trunk and the descending aorta, changes its structure during development. Our previous studies have demonstrated that prostaglandin E2 (PGE2)-EP4 signaling promotes intimal cushion formation (ICF) by activating the migration of DA smooth muscle cells via the secretion of hyaluronan. We hypothesized that, in addition to hyaluronan, PGE2 may secrete other proteins that also regulate vascular remodeling in the DA. In order to detect PGE2 stimulation-secreted proteins, we found that CCN3 protein was increased in the culture supernatant in the presence of PGE2 in a dose-dependent manner by nano-flow liquid chromatography coupled with tandem mass spectrometry analysis and enzyme-linked immunosorbent assay. Quantitative RT-PCR analysis revealed that PGE2 stimulation tended to increase the expression levels of CCN3 mRNA in DA smooth muscle cells. Immunohistochemical analysis revealed that CCN3 was highly localized in the entire smooth muscle layers and the endothelium of the DA. Furthermore, exogenous CCN3 inhibited PGE2-induced ICF in the ex vivo DA tissues. These results suggest that CCN3 is a secreted protein of the DA smooth muscle cells induced by PGE2 to suppress ICF of the DA. The present study indicates that CCN3 could be a novel negative regulator of ICF in the DA to fine-tune the PGE2-mediated DA remodeling.

Truncated dystrophin ameliorates the dystrophic phenotype of mdx mice by reducing sarcolipin-mediated SERCA inhibition.

Duchenne muscular dystrophy (DMD) and the less severe Becker muscular dystrophy (BMD) are due to mutations in the DMD gene. Previous reports show that in-frame deletion of exons 45-55 produces an internally shorted, but functional, dystrophin protein resulting in a very mild BMD phenotype. In order to elucidate the molecular mechanism leading to this phenotype, we generated exon 45-55 deleted dystrophin transgenic/mdx (Tg/mdx) mice. Muscular function of Tg/mdx mice was restored close to that of wild type (WT) mice but the localization of the neuronal type of nitric oxide synthase was changed from the sarcolemma to the cytosol. This led to hyper-nitrosylation of the ryanodine receptor 1 causing increased Ca2+ release from the sarcoplasmic reticulum. On the other hand, Ca2+ reuptake by the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) was restored to the level of WT mice, suggesting that the Ca2+ dysregulation had been compensated by SERCA activation. In line with this, expression of sarcolipin (SLN), a SERCA-inhibitory peptide, was upregulated in mdx mice, but strongly reduced in Tg/mdx mice. Furthermore, knockdown of SLN ameliorated the cytosolic Ca2+ homeostasis and the dystrophic phenotype in mdx mice. These findings suggest that SLN may be a novel target for DMD therapy.

Prostaglandin E2 Receptor EP4 Inhibition Contracts Rat Ductus Arteriosus.

BACKGROUND: Patent ductus arteriosus (PDA) is common in premature infants. Cyclooxygenase inhibitors such as indomethacin, which inhibit prostaglandin E2(PGE2) synthesis, are currently the sole treatments for patients with PDA. Their efficacy are, however, frequently limited, and adverse effects are problematic. Because the PGE2-specific receptor EP4 selectively expresses in rat ductus arteriosus (DA), it is hypothesized that EP4 inhibition would promote DA closure with fewer side-effects.Methods and Results:A new chemical compound EP4 antagonist, RQ-15986 (renamed from CJ-042794), was used. Whether RQ-15986 selectively contracted the DA was examined by measuring the isometric tension of rat DA ex vivo at embryonic day 19 (e19) and e21. RQ-15986 at a dose of 10-6mol/L increased the isometric tension of the DA up to 44.8±6.2% and 69.1±12.9% to the maximal KCl-induced tension at e19 and e21 respectively. The effect of RQ-15986 on rat DA in vivo was also tested by using a rapid whole-body freezing method. RQ-15986 inhibited PGE1-induced DA dilatation in neonatal rats. Furthermore, RQ-15986 contracted the DA in a dose-dependent manner, and the constriction was greater at e21 than at e19. Moreover, RQ-15986 did not contract the aorta or the marginal artery of the colon. CONCLUSIONS: EP4 inhibition contracts rat DA with fewer side-effects. EP4 inhibition is a promising alternative strategy to treat patients with PDA.

Distinct Vascular Remodeling Pattern of Adult Rats with Carotid-Jugular Shunt.

BACKGROUND: Previous research has revealed that patent vein grafts lose their venous identity Eph-B4 but do not gain arterial identity ephrin-B2 during adaptation to the arterial circulation, and vascular identity marker, for example, the Eph-B4 signaling is a critical determinant of venous wall thickness of vein grafts. But what is the remodeling pattern, especially the remodeling pattern of vascular identity in the venous segment of arteriovenous shunt at a late stage postoperation has not been fully explored. This study was conducted to characterize the remodeling pattern of shear stress, vascular identity, structural composition and morphology, and transcriptional profiles in jugular segment of carotid-jugular (CJ) shunt and/or pulmonary artery (PA), which delivers an increased amount of mixed blood at a late stage postoperation in adult rats. METHODS: CJ shunt was created in adult Wistar rats via end-to-end anastomosis of carotid artery (CA) and jugular vein (JV). At the time of 15 weeks, after hemodynamics test, remodeled jugular segment of CJ shunt, PA, and sham-operated corresponding vessels were isolated. Reverse transcription polymerase chain reaction, microarray, western blot, immunohistochemistry experiments, and morphology analyses were performed. RESULTS: CJ shunt shear stresses have been patterned to some sort of balance with no significant difference in shear stress between carotid segment and jugular segment (P > 0.05). Immunohistochemical analysis reveals that venous identity marker Eph-B4 is lost, but arterial identity markers ephrin-B2 and regulator of G-protein signaling 5 are gained in jugular segment of CJ shunt (P < 0.01), and these 2 arterial identity markers further strengthened in PA (P < 0.01) in shunted rats compared with controls. Jugular segment of CJ shunt undergoes significant intimal hyperplasia with strong expression of smooth muscle cell markers (P < 0.05) and demonstrates a distinct transcriptional profiles which reveals that transcripts of 5 arterial markers are significantly upregulated (P < 0.05 or < 0.01) compared with sham-operated JV; among them, G-protein signaling 5 is exactly the gene with the largest fold change (10.14-fold) in all genes tested by microarray experiment. CONCLUSIONS: Venous identity is lost, but arterial identity is gained in jugular segment of CJ shunt and arterial identity further strengthened in PA in adult shunted rats during late adaptation.

Heterozygous deletion of sarcolipin maintains normal cardiac function.

Sarcolipin (SLN) is a small proteolipid and a regulator of sarco(endo)plasmic reticulum Ca(2+)-ATPase. In heart tissue, SLN is exclusively expressed in the atrium. Previously, we inserted Cre recombinase into the endogenous SLN locus by homologous recombination and succeeded in generating SLN-Cre knockin (Sln(Cre/+)) mice. This Sln(Cre/+) mouse can be used to generate an atrium-specific gene-targeting mutant, and it is based on the Cre-loxP system. In the present study, we used adult Sln(Cre/+) mice atria and analyzed the effects of heterozygous SLN deletion by Cre knockin before use as the gene targeting mouse. Both SLN mRNA and protein levels were decreased in Sln(Cre/+) mouse atria, but there were no morphological, physiological, or molecular biological abnormalities. The properties of contractility and Ca(2+) handling were similar to wild-type (WT) mice, and expression levels of several stress markers and sarcoplasmic reticulum-related protein levels were not different between Sln(Cre/+) and WT mice. Moreover, there was no significant difference in sarco(endo)plasmic reticulum Ca(2+)-ATPase activity between the two groups. We showed that Sln(Cre/+) mice were not significantly different from WT mice in all aspects that were examined. The present study provides basic characteristics of Sln(Cre/+) mice and possibly information on the usefulness of Sln(Cre/+) mice as an atrium-specific gene-targeting model.

New mouse model of skeletal muscle atrophy using spiral wire immobilization.

INTRODUCTION: Disuse-induced skeletal muscle atrophy is a serious concern; however, there is not an effective mouse model to elucidate the molecular mechanisms. We developed a noninvasive atrophy model in mice. METHODS: After the ankle joints of mice were bandaged into a bilateral plantar flexed position, either bilateral or unilateral hindlimbs were immobilized by wrapping in bonsai steel wire. RESULTS: After 3, 5, or 10 days of immobilization of the hip, knee, and ankle, the weight of the soleus and plantaris muscles decreased significantly in both bilateral and unilateral immobilization. MAFbx/atrogin-1 and MuRF1 mRNA was found to have significantly increased in both muscles, consistent with disuse-induced atrophy. Notably, the procedure did not result in either edema or necrosis in the fixed hindlimbs. CONCLUSIONS: This method allows repeated, direct access to the immobilized muscle, making it a useful procedure for concurrent application and assessment of various therapeutic interventions. Muscle Nerve 54: 788-791, 2016.

Lipopolysaccharide Delays Closure of the Rat Ductus Arteriosus by Induction of Inducible Nitric Oxide Synthase But Not Prostaglandin E2.

BACKGROUND: The incidence of patent ductus arteriosus is known to be higher in premature neonates with infection than in those without infection. However, the detailed mechanism has not been investigated. METHODS AND RESULTS: Lipopolysaccharide (LPS; 100 µg/kg) was injected into timed-pregnant Wistar rats on day 18 and 19 of pregnancy. The fetuses were delivered by cesarean section on gestational day 21. Using a rapid whole-body freezing method, it was found that closure of the ductus arteriosus (DA) was significantly delayed in neonates from LPS-injected rats after birth. Histological analysis demonstrated that there was no difference in vascular remodeling of the DA. Quantitative reverse transcriptase-polymerase chain reaction analysis showed that the tumor necrosis factor α and inducible nitric oxide synthase (iNOS) mRNA expression level was significantly increased, but there was no difference in cyclooxygenase 2 and prostaglandin receptor, EP4, mRNA expression in the DA from LPS-injected rats. Moreover, the NOS inhibitor,Nω-Nitro-L-arginine methyl ester hydrochloride, significantly prevented the delayed closure of the DA after birth in neonates from LPS-injected rats. CONCLUSIONS: The present study demonstrated that LPS-mediated infection delayed closure of the rat DA without apparent histological changes. iNOS, but not prostaglandin E2, may play a primary role in delayed functional closure of the DA. (Circ J 2016; 80: 703-711).

Prostaglandin E2 inhibits elastogenesis in the ductus arteriosus via EP4 signaling.

BACKGROUND: Elastic fiber formation begins in mid-gestation and increases dramatically during the last trimester in the great arteries, providing elasticity and thus preventing vascular wall structure collapse. However, the ductus arteriosus (DA), a fetal bypass artery between the aorta and pulmonary artery, exhibits lower levels of elastic fiber formation, which promotes vascular collapse and subsequent closure of the DA after birth. The molecular mechanisms for this inhibited elastogenesis in the DA, which is necessary for the establishment of adult circulation, remain largely unknown. METHODS AND RESULTS: Stimulation of the prostaglandin E2 (PGE2) receptor EP4 significantly inhibited elastogenesis and decreased lysyl oxidase (LOX) protein, which catalyzes elastin cross-links in DA smooth muscle cells (SMCs), but not in aortic SMCs. Aortic SMCs expressed much less EP4 than DASMCs. Adenovirus-mediated overexpression of LOX restored the EP4-mediated inhibition of elastogenesis in DASMCs. In EP4-knockout mice, electron microscopic examination showed that the DA acquired an elastic phenotype that was similar to the neighboring aorta. More importantly, human DA and aorta tissues from 7 patients showed a negative correlation between elastic fiber formation and EP4 expression, as well as between EP4 and LOX expression. The PGE2-EP4-c-Src-phospholipase C (PLC)γ-signaling pathway most likely promoted the lysosomal degradation of LOX. CONCLUSIONS: Our data suggest that PGE2 signaling inhibits elastogenesis in the DA, but not in the aorta, through degrading LOX protein. Elastogenesis is spatially regulated by PGE2-EP4 signaling in the DA.

Oxygenation decreases elastin secretion from rat ductus arteriosus smooth muscle cells.

BACKGROUND: The ductus arteriosus (DA), a fetal arterial connection between the main pulmonary artery and the descending aorta, normally closes immediately after birth. The oxygen concentration in the blood rises after birth, and in the DA this increase in oxygen concentration causes functional closure, which is induced by smooth muscle contraction. Previous studies have demonstrated that hypoxia and/or oxygenation affect vascular remodeling of various vessels. Therefore, we hypothesized that the rise in oxygen concentration would affect the vascular structure of the DA due to production of proteins secreted from DA smooth muscle cells (SMC). METHODS AND RESULTS: Liquid chromatography-tandem mass spectrometry was used to comprehensively investigate the secreted proteins in the supernatant of rat DA SMC harvested under hypoxic conditions (1% oxygen) or under normoxic conditions (21% oxygen). We found that the rise in oxygen concentration reduced the secretion of elastin from DA SMC. On reverse transcription-polymerase chain reaction, the expression of elastin mRNA was not significantly changed in DA SMC from hypoxic to normoxic conditions. CONCLUSIONS: Given that elastin forms internal elastic lamina and elastic fibers in the vascular muscle layers, and that a rise in oxygen concentration reduced the secretion of elastin, this suggests that the rise in blood oxygen concentration after birth reduces the secretion of elastin, and therefore may play a role in DA structural remodeling after birth.

Transcription profiles of the ductus arteriosus in Brown-Norway rats with irregular elastic fiber formation.

BACKGROUND: Patent ductus arteriosus (PDA) is one of the most common congenital cardiovascular defects in children. The Brown-Norway (BN) inbred rat presents a higher frequency of PDA. A previous study reported that 2 different quantitative trait loci on chromosomes 8 and 9 were significantly linked to PDA in this strain. Nevertheless, the genetic or molecular mechanisms underlying PDA phenotypes in BN rats have not been fully investigated yet. METHODS AND RESULTS: It was found that the elastic fibers were abundant in the subendothelial area but scarce in the media even in the closed ductus arteriosus (DA) of full-term BN neonates. DNA microarray analysis identified 52 upregulated genes (fold difference >2.5) and 23 downregulated genes (fold difference <0.4) when compared with those of F344 control neonates. Among these genes, 8 (Tbx20, Scn3b, Stac, Sphkap, Trpm8, Rup2, Slc37a2, and RGD1561216) are located in chromosomes 8 and 9. Interestingly, it was also suggested that the significant decrease in the expression levels of the PGE2-specfic receptor, EP4, plays a critical role in elastogenesis in the DA. CONCLUSIONS: BN rats exhibited dysregulation of elastogenesis in the DA. DNA microarray analysis identified the candidate genes including EP4 involved in the DNA phenotype. Further investigation of these newly identified genes will hopefully clarify the molecular mechanisms underlying the irregular formation of elastic fibers in PDA.

αB-Crystallin R120G variant causes cardiac arrhythmias and alterations in the expression of Ca(2+) -handling proteins and endoplasmic reticulum stress in mice.

Mutations of αB-crystallin (CryαB), a small heat shock protein abundantly expressed in cardiac and skeletal muscles, are known to cause desmin-related myopathies. The CryαB R120G allele has been linked to a familial desminopathy and, in transgenic mice, causes a sudden death at about 28 weeks of age. To investigate the mechanisms of the sudden cardiac arrest of CryαB R120G transgenic mice, we prepared protein samples from left ventricular tissues of two different age groups (10 and 28 weeks) and examined Ca(2+) -handling proteins. Expression of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) 2, phospholamban, ryanodine receptor 2 and calsequestrin 2 was significantly decreased in 28- versus 10-week-old CryαB R120G transgenic mice. In addition, low heart rate variability, including heart rate, total power and low frequency, was observed and continuous electrocardiogram monitoring revealed cardiac arrhythmias, such as ventricular tachycardia, atrioventricular block and atrial flutter, in 28-week-old CryαB R120G transgenic mice. In contrast, expression of endoplasmic reticulum (ER) degradation enhancing α-mannosidase-like protein, inositol requirement 1 and X-box binding protein 1 were increased significantly in 28- versus 10-week-old CryαBR120G transgenic mice, suggesting that the CryαBR120G transgenic mice exhibit increased ER stress compared with wild-type mice. Together, the data suggest that the CryαB R120G dominant variant induces ER stress and impairs Ca(2+) regulation, leading to ageing-related cardiac dysfunction, arrhythmias and decreased autonomic tone with shortened lifespan.

Metabolomic profiling analysis reveals chamber-dependent metabolite patterns in the mouse heart.

Energy of the cardiac muscle largely depends on fatty acid oxidation. It is known that the atrium and ventricle have chamber-specific functions, structures, gene expressions, and pathologies. The left ventricle works as a high-pressure chamber to pump blood toward the body, and its muscle wall is thicker than those of the other chambers, suggesting that energy utilization in each of the chambers should be different. However, a chamber-specific pattern of metabolism remains incompletely understood. Recently, innovative techniques have enabled the comprehensive analysis of metabolites. Therefore, we aimed to clarify differences in metabolic patterns among the chambers. Male C57BL6 mice at 6 wk old were subject to a comprehensive measurement of metabolites in the atria and ventricles by capillary electrophoresis and mass spectrometry. We found that overall metabolic profiles, including nucleotides and amino acids, were similar between the right and left ventricles. On the other hand, the atria exhibited a distinct metabolic pattern from those of the ventricles. Importantly, the high-energy phosphate pool (the total concentration of ATP, ADP, and AMP) was higher in both ventricles. In addition, the levels of lactate, acetyl CoA, and tricarboxylic acid cycle contents were higher in the ventricles. Accordingly, the activities and/or expression levels of key enzymes were higher in the ventricles to produce more energy. The present study provides a basis for understanding the chamber-specific metabolism underlining pathophysiology in the heart.

Urinary titin is not an early biomarker of skeletal muscle atrophy induced by muscle denervation in mice.

Early detection of skeletal muscle atrophy is important to prevent further muscle weakness. However, there are few non-invasive biomarkers for skeletal muscle atrophy. Recent studies have reported that the N-terminal fragment (N-titin) of titin, a giant sarcomeric protein, is detected in the urine of patients with muscle damage. In this study, we hypothesized that urinary N-titin would be a potential early biomarker of skeletal muscle atrophy in mice caused by sciatic nerve denervation. Male mice were randomly divided into control and denervation groups, and urinary N-titin levels were assessed daily for 9 days using an enzyme-linked immunosorbent assay system. Despite reduced titin protein levels in atrophic muscles 10 days after denervation, cleaved N-titin fragments were not increased in the urine of mice with denervation-induced muscle atrophy. Furthermore, we found no uptake of Evans blue dye from the extracellular space into the cytoplasm in atrophic muscles, suggesting that the sarcomeric membrane is intact in those muscles. The present results suggest that cleaved N-titin in the urine is not suitable as an early biomarker of skeletal muscle atrophy.

Notch and retinoic acid signals regulate macrophage formation from endocardium downstream of Nkx2-5.

Hematopoietic progenitors are enriched in the endocardial cushion and contribute, in a Nkx2-5-dependent manner, to tissue macrophages required for the remodeling of cardiac valves and septa. However, little is known about the molecular mechanism of endocardial-hematopoietic transition. In the current study, we identified the regulatory network of endocardial hematopoiesis. Signal network analysis from scRNA-seq datasets revealed that genes in Notch and retinoic acid (RA) signaling are significantly downregulated in Nkx2-5-null endocardial cells. In vivo and ex vivo analyses validate that the Nkx2-5-Notch axis is essential for the generation of both hemogenic and cushion endocardial cells, and the suppression of RA signaling via Dhrs3 expression plays important roles in further differentiation into macrophages. Genetic ablation study revealed that these macrophages are essential in cardiac valve remodeling. In summary, the study demonstrates that the Nkx2-5/Notch/RA signaling plays a pivotal role in macrophage differentiation from hematopoietic progenitors.