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.

Thiamine treatment preserves cardiac function against ischemia injury via maintaining mitochondrial size and ATP levels.

Thiamine (vitamin B1) is necessary for energy production, especially in the heart. Recent studies have demonstrated that thiamine supplementation for cardiac diseases is beneficial. However, the detailed mechanisms underlying thiamine-preserved cardiac function have not been elucidated. To this end, we conducted a functional analysis, metabolome analysis, and electron microscopic analysis to unveil the mechanisms of preserved cardiac function through supplementation with thiamine for ischemic cardiac disease. Male Sprague-Dawley rats (around 10 wk old) were used. Following pretreatment with or without thiamine pyrophosphate (TPP; 300 µM), hearts were exposed to ischemia (40 min of global ischemia followed by 60 min of reperfusion). We measured the left ventricle developed pressure (LVDP) throughout the protocol. The LVDP during reperfusion in the TPP-treated heart was significantly higher than that in the untreated heart. Metabolome analysis was performed using capillary electrophoresis-time-of-flight mass spectrometry, and it revealed that the TPP-treated heart retained higher adenosine triphosphate (ATP) levels compared with the untreated heart after ischemia. The metabolic pathway showed that there was a significant increase in fumaric acid and malic acid from the tricarboxylic acid cycle following ischemia. Electron microscope analysis revealed that the mitochondria size in the TPP-treated heart was larger than that in the untreated heart. Mitochondrial fission in the TPP-treated heart was also inhibited, which was confirmed by a decrease in the phosphorylation level of DRP1 (fission related protein). TPP treatment for cardiac ischemia preserved ATP levels probably as a result of maintaining larger mitochondria by inhibiting fission, thereby allowing the TPP-treated heart to preserve contractility performance during reperfusion.NEW & NOTEWORTHY We found that treatment with thiamine can have a protective effect on myocardial ischemia. Thiamine likely mediates mitochondrial fission through the inhibition of DRP1 phosphorylation and the preservation of larger-sized mitochondria and ATP concentration, leading to higher cardiac contractility performance during the subsequent reperfusion state.

Fibrosis growth factor 23 is a promoting factor for cardiac fibrosis in the presence of transforming growth factor-ß1.

Myocardial fibrosis is often associated with cardiac hypertrophy; indeed, fibrosis is one of the most critical factors affecting prognosis. We aimed to identify the molecules involved in promoting fibrosis under hypertrophic stimuli. We previously established a rat model of cardiac hypertrophy by pulmonary artery banding, in which approximately half of the animals developed fibrosis in the right ventricle. Here, we first comprehensively analyzed mRNA expression in the right ventricle with or without fibrosis in pulmonary artery banding model rats by DNA microarray analysis (GSE141650 at NCBI GEO). The expression levels of 19 genes were up-regulated more than 1.5-fold in fibrotic hearts compared with non-fibrotic hearts. Among them, fibrosis growth factor (FGF) 23 showed one of the biggest increases in expression. Real-time PCR analysis also revealed that, among the FGF receptor (FGFR) family, FGFR1 was highly expressed in fibrotic hearts. We then found that FGF23 was expressed predominantly in cardiomyocytes, while FGFR1 was predominantly expressed in fibroblasts in the rat ventricle. Next, we added FGF23 and transforming growth factor (TGF)-ß1 (10-50 ng/mL of each) to isolated fibroblasts from normal adult rat ventricles and cultured them for three days. While FGF23 itself did not directly affect the expression levels of any fibrosis-related mRNAs, FGF23 enhanced the effect of TGF-ß1 on increasing the expression levels of α-smooth muscle actin (α-SMA) mRNA. This increase in xx-SMA mRNA levels due to the combination of TGF-ß1 and FGF23 was attenuated by the inhibition of FGFR1 or the knockdown of FGFR1 in fibroblasts. Thus, FGF23 synergistically promoted the activation of fibroblasts with TGF-ß1, transforming fibroblasts into myofibroblasts via FGFR1. Thus, we identified FGF23 as a paracrine factor secreted from cardiomyocytes to promote cardiac fibrosis under conditions in which TGF-ß1 is activated. FGF23 could be a possible target to prevent fibrosis following myocardial hypertrophy.

Effect of Long-term Administration of Prostaglandin E1 on Morphologic Changes in Ductus Arteriosus.

BACKGROUND: To improve survival of patients with hypoplastic left heart syndrome, combination therapy with bilateral pulmonary artery banding and prostaglandin E1 (PGE1)-mediated ductal patency was developed as an alternative for high-risk neonates in Japan. However, the effect of long-term PGE1 administration on ductus arteriosus remains unclear. Synchrotron radiation-based X-ray phase-contrast tomography (XPCT) enables clear visualization of soft tissues at an approximate spatial resolution of 12.5 µm. We aimed to investigate morphologic changes in ductus arteriosus after long-term PGE1 infusion using XPCT. METHODS: Seventeen ductus arteriosus tissue samples from patients with hypoplastic left heart syndrome were obtained during the Norwood procedure. The median duration of lipo-prostaglandin E1 (lipo-PGE1) administration was 48 days (range, 3 to 123). Structural analysis of ductus arteriosus was performed and compared with conventional histologic analysis. RESULTS: The XPCT was successfully applied to quantitative measurements of ductal media. Significant correlation was found between the duration of lipo-PGE1 infusion and mass density of ductal media (R = 0.723, P = .001). The duration of lipo-PGE1 administration was positively correlated with elastic fiber staining (R = 0.799, P < .001) and negatively correlated with smooth muscle formation (R = -0.83, P < .001). No significant increase in intimal cushion formation was found after long-term lipo-PGE1 administration. Expression of ductus arteriosus dominant PGE2-receptor EP4 almost disappeared in specimens when lipo-PGE1 was administered over 3 days. CONCLUSIONS: Disorganized elastogenesis and little intimal cushion formation after long-term lipo-PGE1 administration suggest that ductus arteriosus remodeled to the elastic artery phenotype. Because EP4 was downregulated and ductus arteriosus exhibited elastic characteristics, the dosage of lipo-PGE1 might be decreased after a definite administration period.

Standard-dose gentamicin does not increase risk of patent ductus arteriosus.

BACKGROUND: Rates of patent ductus arteriosus (PDA) and infection are high in preterm infants. Preterm infants with infection are more likely to develop symptomatic PDA, a potentially fatal disease. Clinically, gentamicin is widely used for early-onset infection in neonates including preterm infants. A recent study demonstrated that standard-dose gentamicin itself, not infection, increased risk of PDA in mice, suggesting that gentamicin should be avoided in neonates with a risk of PDA. This claim has been insufficiently investigated in subsequent in-vivo experiments. We reevaluated the in-vivo effect of standard-dose gentamicin on patency of the rat ductus arteriosus (DA). METHODS: 1) To evaluate the effect of gentamicin on DA patency duration, gentamicin was intraperitoneally injected immediately after birth. 2) To evaluate the effect of gentamicin on DA reopening, gentamicin was intraperitoneally injected 30 min after birth. In both scenarios, 30 min after gentamicin administration, rapid whole-body freezing was performed and the inner diameter of the DA was measured. RESULTS: Standard-dose gentamicin (5 µg/g) did not prolong patency of the DA or increase the likelihood of DA reopening in rat neonates. High-dose gentamicin (100 µg/g), however, significantly prolonged patency of the DA and was associated with DA reopening in rat neonates, although the dilative effect did not reach statistical significance. CONCLUSION: Standard-dose gentamicin does not increase the risk of PDA in rat neonates. This study suggests that standard-dose gentamicin can be used to treat infection in neonates without increasing PDA morbidity.

Transcriptional profiles in the chicken ductus arteriosus during hatching.

The ductus arteriosus, an essential embryonic blood vessel between the pulmonary artery and the descending aorta, constricts after birth or hatching and eventually closes to terminate embryonic circulation. Chicken embryos have two long ductus arteriosi, which anatomically differ from mammal ductus arteriosus. Each long ductus arteriosus is divided into two parts: the pulmonary artery-sided and descending aorta-sided ductus arteriosi. Although the pulmonary artery-sided and descending aorta-sided ductus arteriosi have distinct functional characteristics, such as oxygen responsiveness, the difference in their transcriptional profiles has not been investigated. We performed a DNA microarray analysis (GSE 120116 at NCBI GEO) with pooled tissues from the chicken pulmonary artery-sided ductus arteriosus, descending aorta-sided ductus arteriosus, and aorta at the internal pipping stage. Although several known ductus arteriosus-dominant genes such as tfap2b were highly expressed in the pulmonary artery-sided ductus arteriosus, we newly found genes that were dominantly expressed in the chicken pulmonary artery-sided ductus arteriosus. Interestingly, cluster analysis showed that the expression pattern of the pulmonary artery-sided ductus arteriosus was closer to that of the descending aorta-sided ductus arteriosus than that of the aorta, whereas the morphology of the descending aorta-sided ductus arteriosus was closer to that of the aorta than that of the pulmonary artery-sided ductus arteriosus. Subsequent pathway analysis with DAVID bioinformatics resources revealed that the pulmonary artery-sided ductus arteriosus showed enhanced expression of the genes involved in melanogenesis and tyrosine metabolism compared with the descending aorta-sided ductus arteriosus, suggesting that tyrosinase and the related genes play an important role in the proper differentiation of neural crest-derived cells during vascular remodeling in the ductus arteriosus. In conclusion, the transcription profiles of the chicken ductus arteriosus provide new insights for investigating the mechanism of ductus arteriosus closure.

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.

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.

A Sarcoplasmic Reticulum Localized Protein Phosphatase Regulates Phospholamban Phosphorylation and Promotes Ischemia Reperfusion Injury in the Heart.

Phospholamban (PLN) is a key regulator of sarcolemma calcium uptake in cardiomyocyte, its inhibitory activity to SERCA is regulated by phosphorylation. PLN hypophosphorylation is a common molecular feature in failing heart. The current study provided evidence at molecular, cellular and whole heart levels to implicate a sarcolemma membrane targeted protein phosphatase, PP2Ce, as a specific and potent PLN phosphatase. PP2Ce expression was elevated in failing human heart and induced acutely at protein level by ß -adrenergic stimulation or oxidative stress in cardiomyocytes. PP2Ce expression in mouse heart blunted ß-adrenergic response and exacerbated ischemia/reperfusion injury. Therefore, PP2Ce is a new regulator for cardiac function and pathogenesis.

Pulmonary hypertension due to left heart disease causes intrapulmonary venous arterialization in rats.

OBJECTIVE: A rat model of left atrial stenosis-associated pulmonary hypertension due to left heart diseases was prepared to elucidate its mechanism. METHODS: Five-week-old Sprague-Dawley rats were randomly divided into 2 groups: left atrial stenosis and sham-operated control. Echocardiography was performed 2, 4, 6, and 10 weeks after surgery, and cardiac catheterization and organ excision were subsequently performed at 10 weeks after surgery. RESULTS: Left ventricular inflow velocity, measured by echocardiography, significantly increased in the left atrial stenosis group compared with that in the sham-operated control group (2.2 m/s, interquartile range [IQR], 1.9-2.2 and 1.1 m/s, IQR, 1.1-1.2, P < .01), and the right ventricular pressure-to-left ventricular systolic pressure ratio significantly increased in the left atrial stenosis group compared with the sham-operated control group (0.52, IQR, 0.54-0.60 and 0.22, IQR, 0.15-0.27, P < .01). The right ventricular weight divided by body weight was significantly greater in the left atrial stenosis group than in the sham-operated control group (0.54 mg/g, IQR, 0.50-0.59 and 0.39 mg/g, IQR, 0.38-0.43, P < .01). Histologic examination revealed medial hypertrophy of the pulmonary vein was thickened by 1.6 times in the left atrial stenosis group compared with the sham-operated control group. DNA microarray analysis and real-time polymerase chain reaction revealed that transforming growth factor-ß mRNA was significantly elevated in the left atrial stenosis group. The protein levels of transforming growth factor-ß and endothelin-1 were increased in the lung of the left atrial stenosis group by Western blot analyses. CONCLUSIONS: We successfully established a novel, feasible rat model of pulmonary hypertension due to left heart diseases by generating left atrial stenosis. Although pulmonary hypertension was moderate, the pulmonary hypertension due to left heart diseases model rats demonstrated characteristic intrapulmonary venous arterialization and should be used to further investigate the mechanism of pulmonary hypertension due to left heart diseases.

Impairment of Excitation-Contraction Coupling in Right Ventricular Hypertrophied Muscle with Fibrosis Induced by Pulmonary Artery Banding.

Interstitial myocardial fibrosis is one of the factors responsible for dysfunction of the heart. However, how interstitial fibrosis affects cardiac function and excitation-contraction coupling (E-C coupling) has not yet been clarified. We developed an animal model of right ventricular (RV) hypertrophy with fibrosis by pulmonary artery (PA) banding in rats. Two, four, and six weeks after the PA-banding operation, the tension and intracellular Ca2+ concentration of RV papillary muscles were simultaneously measured (n = 33). The PA-banding rats were clearly divided into two groups by the presence or absence of apparent interstitial fibrosis in the papillary muscles: F+ or F- group, respectively. The papillary muscle diameter and size of myocytes were almost identical between F+ and F-, although the RV free wall weight was heavier in F+ than in F-. F+ papillary muscles exhibited higher stiffness, lower active tension, and lower Ca2+ responsiveness compared with Sham and F- papillary muscles. In addition, we found that the time to peak Ca2+ had the highest correlation coefficient to percent of fibrosis among other parameters, such as RV weight and active tension of papillary muscles. The phosphorylation level of troponin I in F+ was significantly higher than that in Sham and F-, which supports the idea of lower Ca2+ responsiveness in F+. We also found that connexin 43 in F+ was sparse and disorganized in the intercalated disk area where interstitial fibrosis strongly developed. In the present study, the RV papillary muscles obtained from the PA-banding rats enabled us to directly investigate the relationship between fibrosis and cardiac dysfunction, the impairment of E-C coupling in particular. Our results suggest that interstitial fibrosis worsens cardiac function due to 1) the decrease in Ca2+ responsiveness and 2) the asynchronous activation of each cardiac myocyte in the fibrotic preparation due to sparse cell-to-cell communication.

Tissue thrombin is associated with the pathogenesis of dilated cardiomyopathy.

BACKGROUND: Thrombin is a serine protease known to be the final product of the coagulation cascade. However, thrombin plays other physiological roles in processes such as gastric contractions and vessel wound healing, and a state of coagulability is increased in patients with dilated cardiomyopathy (DCM). In this study, we investigate the role of thrombin in the pathogenesis of DCM. The purpose of this study is to clarify the role of thrombin in the pathogenesis of DCM and investigate the possibility of treatment against DCM by thrombin inhibition. METHODS: We investigated the expression of thrombin in the left ventricles of five patients with DCM who underwent the Batista operation and four patients without heart disease. Furthermore, we investigated the involvement of thrombin in the development of DCM using knock-in mice with a deletion mutation of cardiac troponin T that causes human DCM (∆K210 knock-in mouse) (B6;129-Tnnt2tm2Mmto) and assessed the effects of a direct thrombin inhibitor, dabigatran on ∆K210 knock-in mice using echocardiographic examinations, the Kaplan-Meier method and Western blotting. RESULTS: The immunohistochemical analysis showed a strong thrombin expression in the DCM patients compared to the patients without heart disease. In immunohistochemical analysis, a strong thrombin expression was observed in the heart tissues analysis in the ∆K210 knock-in mice. Dabigatran administration significantly improved fractional shortening according to the echocardiographic examination and the survival outcomes in ∆K210 knock-in mice. CONCLUSION: Tissue thrombin is involved in the pathogenesis of DCM and thrombin inhibition can be beneficial for the treatment of DCM.

Low Cardiac Output Leads Hepatic Fibrosis in Right Heart Failure Model Rats.

BACKGROUND: Hepatic fibrosis progresses with right heart failure, and becomes cardiac cirrhosis in a severe case. Although its causal factor still remains unclear. Here we evaluated the progression of hepatic fibrosis using a pulmonary artery banding (PAB)-induced right heart failure model and investigated whether cardiac output (CO) is responsible for the progression of hepatic fibrosis. METHODS AND RESULTS: Five-week-old Sprague-Dawley rats divided into the PAB and sham-operated control groups. After 4 weeks from operation, we measured CO by echocardiography, and hepatic fibrosis ratio by pathological examination using a color analyzer. In the PAB group, CO was significantly lower by 48% than that in the control group (78.2±27.6 and 150.1±31.2 ml/min, P<0.01). Hepatic fibrosis ratio and serum hyaluronic acid, an index of hepatic fibrosis, were significantly increased in the PAB group than those in the control group (7.8±1.7 and 1.0±0.2%, P<0.01, 76.2±27.5 and 32.7±7.5 ng/ml, P<0.01). Notably, the degree of hepatic fibrosis significantly correlated a decrease in CO. Immunohistological analysis revealed that hepatic stellate cells were markedly activated in hypoxic areas, and HIF-1α positive hepatic cells were increased in the PAB group. Furthermore, by real-time PCR analyses, transcripts of profibrotic and fibrotic factors (TGF-ß1, CTGF, procollargen I, procollargen III, MMP 2, MMP 9, TIMP 1, TIMP 2) were significantly increased in the PAB group. In addition, western blot analyses revealed that the protein level of HIF-1α was significantly increased in the PAB group than that in the control group (2.31±0.84 and 1.0±0.18 arbitrary units, P<0.05). CONCLUSIONS: Our study demonstrated that low CO and tissue hypoxia were responsible for hepatic fibrosis in right failure heart model rats.

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).

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.

T-type Ca2+ channels promote oxygenation-induced closure of the rat ductus arteriosus not only by vasoconstriction but also by neointima formation.

The ductus arteriosus (DA), an essential vascular shunt for fetal circulation, begins to close immediately after birth. Although Ca(2+) influx through several membrane Ca(2+) channels is known to regulate vasoconstriction of the DA, the role of the T-type voltage-dependent Ca(2+) channel (VDCC) in DA closure remains unclear. Here we found that the expression of alpha1G, a T-type isoform that is known to exhibit a tissue-restricted expression pattern in the rat neonatal DA, was significantly up-regulated in oxygenated rat DA tissues and smooth muscle cells (SMCs). Immunohistological analysis revealed that alpha1G was localized predominantly in the central core of neonatal DA at birth. DA SMC migration was significantly increased by alpha1G overexpression. Moreover, it was decreased by adding alpha1G-specific small interfering RNAs or using R(-)-efonidipine, a highly selective T-type VDCC blocker. Furthermore, an oxygenation-mediated increase in an intracellular Ca(2+) concentration of DA SMCs was significantly decreased by adding alpha1G-specific siRNAs or using R(-)-efonidipine. Although a prostaglandin E receptor EP4 agonist potently promoted intimal thickening of the DA explants, R(-)-efonidipine (10(-6) m) significantly inhibited EP4-promoted intimal thickening by 40% using DA tissues at preterm in organ culture. Moreover, R(-)-efonidipine (10(-6) m) significantly attenuated oxygenation-induced vasoconstriction by approximately 27% using a vascular ring of fetal DA at term. Finally, R(-)-efonidipine significantly delayed the closure of in vivo DA in neonatal rats. These results indicate that T-type VDCC, especially alpha1G, which is predominantly expressed in neonatal DA, plays a unique role in DA closure, implying that T-type VDCC is an alternative therapeutic target to regulate the patency of DA.

Sarcalumenin is essential for maintaining cardiac function during endurance exercise training.

Sarcalumenin (SAR), a Ca(2+)-binding protein located in the longitudinal sarcoplasmic reticulum (SR), regulates Ca(2+) reuptake into the SR by interacting with cardiac sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a). We have previously demonstrated that SAR deficiency induced progressive heart failure in response to pressure overload, despite mild cardiac dysfunction in sham-operated SAR knockout (SARKO) mice (26). Since responses to physiological stresses often differ from those to pathological stresses, we examined the effects of endurance exercise on cardiac function in SARKO mice. Wild-type (WT) and SARKO mice were subjected to endurance treadmill exercise training ( approximately 65% of maximal exercise ability for 60 min/day) for 12 wk. After exercise training, maximal exercise ability was significantly increased by 5% in WT mice (n = 6), whereas it was significantly decreased by 37% in SARKO mice (n = 5). Cardiac function assessed by echocardiographic examination was significantly decreased in accordance with upregulation of biomarkers of cardiac stress in SARKO mice after training. After training, expression levels of SERCA2a protein were significantly downregulated by 30% in SARKO hearts, whereas they were significantly upregulated by 59% in WT hearts. Consequently, SERCA2 activity was significantly decreased in SARKO hearts after training. Furthermore, the expression levels of other Ca(2+)-handling proteins, including phospholamban, ryanodine receptor 2, calsequestrin 2, and sodium/calcium exchanger 1, were significantly decreased in SARKO hearts after training. These results indicate that SAR plays a critical role in maintaining cardiac function under physiological stresses, such as endurance exercise, by regulating Ca(2+) transport activity into the SR. SAR may be a primary target for exercise-related adaptation of the Ca(2+) storage system in the SR to preserve cardiac function.

Comparison of markers for fetal inflammatory response syndrome: fetal blood interleukin-6 and neonatal urinary beta(2)-microglobulin.

AIM: Chronic lung disease (CLD) is a major component in the morbidity of premature infants suffering from fetal inflammatory response (FIRS). The aim of the present study was to compare the value of measuring neonatal urinary beta(2)-microglobulin (beta(2)-MG) levels with fetal blood interleukin (IL)-6 levels in premature infants at risk of developing CLD. METHODS: Premature infants (gestational age <30 weeks) without CLD (n = 19) and with CLD (n = 10) were enrolled. We measured IL-6 levels in umbilical cord blood and beta(2)-MG levels in urine obtained within 48 h after birth. RESULTS: IL-6 and beta(2)-MG levels were significantly higher in infants who developed CLD than in those who did not (median IL-6, 54.7 vs 7.6 pg/mL; P < 0.005; beta(2)-MG 17.7 vs 9.3 x 10(4) microg/gCr; P < 0.05). The sensitivity and negative predictive value of beta(2)-MG at the cut-off value at 10.0 x 10(4) microg/gCr (0.90 and 0.92) were comparable to IL-6 at 16 pg/mL (0.90 and 0.94). CONCLUSION: We suggest that measuring urinary beta(2)-MG in premature infants soon after birth can monitor FIRS and may provide information on the risk of subsequent CLD development that is as clinically important as information derived from umbilical cord blood IL-6.

Long-term electrocardiographic follow-up from childhood of an adult patient with Brugada syndrome associated with sick sinus syndrome.

We had the unique opportunity of following the electrocardiographic (ECG) course of a 13-year-old male with sinus dysfunction and atrial flutter who subsequently developed a Brugada-type ECG pattern associated with sick sinus syndrome at 25 years old. Family history showed that the patient's mother and maternal grandfather suddenly died while sleeping at night. When the patient was 13 years old, he lost consciousness after running a marathon. The patient was diagnosed with sinus dysfunction and atrial flutter, and he underwent pacemaker implantation at 15 years old. ECG examinations performed between 13 and 20 years old showed incomplete right bundle branch block and ST elevation with early depolarization. On ECG examinations performed when the patient was 21 years old and thereafter, the V(2) lead always showed a saddleback-type ST elevation. At 25 years old, the late potential was positive and the electrophysiological study induced ventricular fibrillation. A challenge test with pilsicainide showed remarkable ST elevation by the V(2) lead. The 24-h Holter ECG monitoring showed remarkable ST elevation after eating a snack and during night time when the patient was asleep. The patient was diagnosed with Brugada syndrome and an implantable cardioverter-defibrillator was implanted. Genetic analysis did not reveal mutation of the SCN5A gene.

Prostaglandin E2-activated Epac promotes neointimal formation of the rat ductus arteriosus by a process distinct from that of cAMP-dependent protein kinase A.

We have demonstrated that chronic stimulation of the prostaglandin E2-cAMP-dependent protein kinase A (PKA) signal pathway plays a critical role in intimal cushion formation in perinatal ductus arteriosus (DA) through promoting synthesis of hyaluronan. We hypothesized that Epac, a newly identified effector of cAMP, may play a role in intimal cushion formation (ICF) in the DA distinct from that of PKA. In the present study, we found that the levels of Epac1 and Epac2 mRNAs were significantly up-regulated in the rat DA during the perinatal period. A specific EP4 agonist, ONO-AE1-329, increased Rap1 activity in the presence of a PKA inhibitor, PKI-(14-22)-amide, in DA smooth muscle cells. 8-pCPT-2'-O-Me-cAMP (O-Me-cAMP), a cAMP analog selective to Epac activator, promoted migration of DA smooth muscle cells (SMC) in a dose-dependent manner. Adenovirus-mediated Epac1 or Epac2 gene transfer further enhanced O-Me-cAMP-induced cell migration, although the effect of Epac1 overexpression on cell migration was stronger than that of Epac2. In addition, transfection of small interfering RNAs for Epac1, but not Epac2, significantly inhibited serum-mediated migration of DA SMCs. In the presence of O-Me-cAMP, actin stress fibers were well organized with enhanced focal adhesion, and cell shape was widely expanded. Adenovirus-mediated Epac1, but not Epac2 gene transfer, induced prominent ICF in the rat DA explants when compared with those with green fluorescent protein gene transfer. The thickness of intimal cushion became significantly greater (1.98-fold) in Epac1-overexpressed DA. O-Me-cAMP did not change hyaluronan production, although it decreased proliferation of DA SMCs. The present study demonstrated that Epac, especially Epac1, plays an important role in promoting SMC migration and thereby ICF in the rat DA.