Comparison of the electrophysiological properties of the pulmonary veins between paroxysmal and persistent atrial fibrillation.

Background: The role of the pulmonary veins (PVs) as triggers in atrial fibrillation (AF) is well-known; however, their detailed electrophysiological properties have not been thoroughly examined. Objective: This study aimed to investigate the electrophysiological properties of the PVs between paroxysmal AF (pAF) and persistent AF (perAF). Methods: Prior to catheter ablation in patients with pAF (n = 51) and perAF (n = 41), a voltage map of the left atrium and PVs was created under sinus rhythm, and the area of the myocardial sleeves in the PVs and their electrophysiological characteristics, including the pacing threshold and effective refractory period (ERP), were compared between the two groups. Results: Compared with perAF, the myocardial sleeves of PVs for pAF were significantly larger for all PVs. The ERP for perAF was significantly shorter than that for pAF for all PVs. The pacing threshold for perAF was significantly higher than that for pAF for the right and left superior PVs. Conclusion: In patients with perAF, a decrease in the normal myocardial sleeves and a shortening of the ERP were observed for all PVs. Those changes in the electrophysiological properties of the PVs might be related to the persistence of AF.

Reactive Oxygen Species in the Aorta and Perivascular Adipose Tissue Precedes Endothelial Dysfunction in the Aorta of Mice with a High-Fat High-Sucrose Diet and Additional Factors.

Metabolic syndrome (Mets) is the major contributor to the onset of metabolic complications, such as hypertension, type 2 diabetes mellitus (DM), dyslipidemia, and non-alcoholic fatty liver disease, resulting in cardiovascular diseases. C57BL/6 mice on a high-fat and high-sucrose diet (HFHSD) are a well-established model of Mets but have minor endothelial dysfunction in isolated aortas without perivascular adipose tissue (PVAT). The purpose of this study was to evaluate the effects of additional factors such as DM, dyslipidemia, and steatohepatitis on endothelial dysfunction in aortas without PVAT. Here, we employed eight-week-old male C57BL/6 mice fed with a normal diet (ND), HFHSD, steatohepatitis choline-deficient HFHSD (HFHSD-SH), and HFHSD containing 1% cholesterol and 0.1% deoxycholic acid (HFHSD-Chol) for 16 weeks. At week 20, some HFHSD-fed mice were treated with streptozocin to develop diabetes (HFHSD-DM). In PVAT-free aortas, the endothelial-dependent relaxation (EDR) did not differ between ND and HFHSD (p = 0.25), but in aortas with PVAT, the EDR of HFHSD-fed mice was impaired compared with ND-fed mice (p = 0.005). HFHSD-DM, HFHSD-SH, and HFHSD-Chol impaired the EDR in aortas without PVAT (p < 0.001, p = 0.019, and p = 0.009 vs. ND, respectively). Furthermore, tempol rescued the EDR in those models. In the Mets model, the EDR is compromised by PVAT, but with the addition of DM, dyslipidemia, and SH, the vessels themselves may result in impaired EDR.

Endothelial Extracellular Signal-Regulated Kinase/Thromboxane A2/Prostanoid Receptor Pathway Aggravates Endothelial Dysfunction and Insulin Resistance in a Mouse Model of Metabolic Syndrome.

Background Metabolic syndrome is characterized by insulin resistance, which impairs intracellular signaling pathways and endothelial NO bioactivity, leading to cardiovascular complications. Extracellular signal-regulated kinase (ERK) is a major component of insulin signaling cascades that can be activated by many vasoactive peptides, hormones, and cytokines that are elevated in metabolic syndrome. The aim of this study was to clarify the role of endothelial ERK2 in vivo on NO bioactivity and insulin resistance in a mouse model of metabolic syndrome. Methods and Results Control and endothelial-specific ERK2 knockout mice were fed a high-fat/high-sucrose diet (HFHSD) for 24 weeks. Systolic blood pressure, endothelial function, and glucose metabolism were investigated. Systolic blood pressure was lowered with increased NO products and decreased thromboxane A2/prostanoid (TP) products in HFHSD-fed ERK2 knockout mice, and Nω-nitro-l-arginine methyl ester (L-NAME) increased it to the levels observed in HFHSD-fed controls. Acetylcholine-induced relaxation of aortic rings was increased, and aortic superoxide level was lowered in HFHSD-fed ERK2 knockout mice. S18886, an antagonist of the TP receptor, improved endothelial function and decreased superoxide level only in the rings from HFHSD-fed controls. Glucose intolerance and the impaired insulin sensitivity were blunted in HFHSD-fed ERK2 knockout mice without changes in body weight. In vivo, S18886 improved endothelial dysfunction, systolic blood pressure, fasting serum glucose and insulin levels, and suppressed nonalcoholic fatty liver disease scores only in HFHSD-fed controls. Conclusions Endothelial ERK2 increased superoxide level and decreased NO bioactivity, resulting in the deterioration of endothelial function, insulin resistance, and steatohepatitis, which were improved by a TP receptor antagonist, in a mouse model of metabolic syndrome.

Metabolic Remodeling with Hepatosteatosis Induced Vascular Oxidative Stress in Hepatic ERK2 Deficiency Mice with High Fat Diets.

We previously demonstrated the marked hepatosteatosis and endothelial dysfunction in hepatocyte-specific ERK2 knockout mice (LE2KO) with a high-fat/high-sucrose diet (HFHSD), but detailed metabolic changes and the characteristics in insulin-sensitive organs were not tested. This study aimed to characterize metabolic remodeling with changes in insulin-sensitive organs, which could induce endothelial dysfunction in HFHSD-LE2KO. The serum glucose and fatty acid (FA) were modestly higher in HFHSD-LE2KO than HFHSD-Control. FA synthesis genes were up-regulated, which was associated with the decreased phosphorylation of AMPK and ACC, and with the up-regulation of SREBP-1 in the liver from HFHSD-LE2KO. In FA and amino acids fraction analysis, arachidonic acid/eicosapentaenoic acid ratio, L-ornithine/arginine ratio, asymmetric dimethylarginine and homocysteine levels were elevated in HFHSD-LE2KO. Insulin-induced phosphorylation of AKT was blunted in skeletal muscle. Serum leptin and IL-1ß were elevated, and serum adiponectin was decreased with the enlargement of epididymal adipocytes. Finally, the enhanced superoxide levels in the aorta, which were blunted with CCCP, apocynin, and tempol, were observed in HFHSD-LE2KO. A pre-incubation of aortic rings with tempol improved endothelial dysfunction in HFHSD-LE2KO. HFHSD-LE2KO revealed an acceleration of FA synthesis in the liver leading to insulin resistance in skeletal muscle and the enlargement of visceral adipocytes. Global metabolic remodeling such as changes in arginine metabolism, ω3/ω6 ratio, and adipocytokines, could affect the vascular oxidative stress and endothelial dysfunction in HFHSD-LE2KO.

Resistance to Obesity in SOD1 Deficient Mice with a High-Fat/High-Sucrose Diet.

Metabolic syndrome (Mets) is an important condition because it may cause stroke and heart disease in the future. Reactive oxygen species (ROSs) influence the pathogenesis of Mets; however, the types of ROSs and their localization remain largely unknown. In this study, we investigated the effects of SOD1, which localize to the cytoplasm and mitochondrial intermembrane space and metabolize superoxide anion, on Mets using SOD1 deficient mice (SOD1-/-). SOD1-/- fed on a high-fat/high-sucrose diet (HFHSD) for 24 weeks showed reduced body weight gain and adipose tissue size compared to wild-type mice (WT). Insulin secretion was dramatically decreased in SOD1-/- fed on HFHSD even though blood glucose levels were similar to WT. Ambulatory oxygen consumption was accelerated in SOD1-/- with HFHSD; however, ATP levels of skeletal muscle were somewhat reduced compared to WT. Reflecting the reduced ATP, the expression of phosphorylated AMPK (Thr 172) was more robust in SOD1-/-. SOD1 is involved in the ATP production mechanism in mitochondria and may contribute to visceral fat accumulation by causing insulin secretion and insulin resistance.

Sarco/Endoplasmic Reticulum Ca2+ ATPase 2 Activator Ameliorates Endothelial Dysfunction; Insulin Resistance in Diabetic Mice.

Background: Sarco/endoplasmic reticulum Ca2+-ATPase2 (SERCA2) is impaired in various organs in animal models of diabetes. The purpose of this study was to test the effects of an allosteric SERCA2 activator (CDN1163) on glucose intolerance, hepatosteatosis, skeletal muscle function, and endothelial dysfunction in diabetic (db/db) mice. Methods: Either CDN1163 or vehicle was injected intraperitoneally into 16-week-old male control and db/db mice for 5 consecutive days. Results: SERCA2 protein expression was decreased in the aorta of db/db mice. In isometric tension measurements of aortic rings from db/db mice treated with CDN1163, acetylcholine (ACh)-induced relaxation was improved. In vivo intraperitoneal administrations of CDN 1163 also increased ACh-induced relaxation. Moreover, CDN1163 significantly decreased blood glucose in db/db mice at 60 and 120 min during a glucose tolerance test; it also decreased serum insulin levels, hepatosteatosis, and oxygen consumption in skeletal muscle during the early period of exercise in db/db mice. Conclusions: CDN1163 directly improved aortic endothelial dysfunction in db/db mice. Moreover, CDN1163 improved hepatosteatosis, skeletal muscle function, and insulin resistance in db/db mice. The activation of SERCA2 might be a strategy for the all the tissue expressed SERCA2a improvement of endothelial dysfunction and the target for the organs related to insulin resistance.

Artificially Created Reentry Circuit by Laser Irradiation Causes Atrial Tachycardia to Persist in Murine Atria.

BACKGROUND: There is a gradual progression from paroxysmal to persistent atrial fibrillation (AF) in humans. To elucidate the mechanism involved, the creation of an artificial atrial substrate to persist AF in mice was attempted.Methods and Results:This study used wild type (WT) mice, but it is difficult to induce AF in them. A novel antegrade perfusion method from the left ventricle (LV) to enlarge both atria for artificial atrial modification was proposed in this study. Short duration AF was induced by burst pacing under this method. Optical mapping analysis revealed non-sustained focal type and meandering spiral reentrants after short duration AF. A tiny artificial substrate (~1.2 mm in diameter) was added in by laser irradiation to create a critical atrial arrhythmogenic substrate. Burst pacing was performed in a non-laser group (n=8), a circular-shape laser group (n=8), and a wedge-shaped dent laser group (n=8). We defined AF and atrial tachycardia (AT) as atrial arrhythmia (AA). Long-lasting AA was defined as lasting for ≥30 min. Long-lasting AA was observed in 0/8, 0/8, and 6/8 (75%) mice in each group. Optical mapping analysis revealed that the mechanism was AT with a stationary rotor around the irradiated margin. CONCLUSIONS: Regrettably, this study failed to reproduce persistent AF, but succeeded in creating an arrhythmic substrate that causes sustained AT in WT mice.

Impact of oxidative posttranslational modifications of SERCA2 on heart failure exacerbation in young patients with non-ischemic cardiomyopathy: A pilot study.

BACKGROUND: Oxidative posttranslational modifications (OPTM) impair the function of Sarcoplasmic/endoplasmic reticulum (SR) calcium (Ca2+) ATPase (SERCA) 2 and trigger cytosolic Ca2+ dysregulation. We investigated the extent of OPTM of SERCA2 in patients with non-ischemic cardiomyopathy (NICM). METHODS AND RESULTS: Endomyocardial biopsy (EMB) was obtained in 40 consecutive patients with NICM. Total expression and OPTM of SERCA2, including sulfonylation at cysteine-674 (S-SERCA2) and nitration at tyrosine-294/295 (N-SERCA2), were examined by immunohistochemical analysis. S-SERCA2 increased in the presence of late gadolinium enhancement on cardiac magnetic resonance imaging. S-SERCA2/SERCA2 and N-SERCA2/SERCA2 correlated with cardiac fibrosis evaluated by Masson's trichrome staining of EMB. SERCA2 expression modestly increased in parallel with an upward trend in OPTM of SERCA2 with aging. This tendency became prominent only in patients aged >65 years. OPTM of SERCA2 positively correlated with brain natriuretic peptide (BNP) values only in patients aged ≤65 years. Composite major adverse cardiac events (MACE) increased more in the high OPTM group of younger patients; however, MACE-free survival was similar irrespective of the extent of OPTM in older patients. CONCLUSIONS: OPTM of SERCA2 correlate with myocardial fibrosis in NICM. In younger patients, OPTM of SERCA2 correlate with elevated BNP and increased composite MACE.

Production of adeno-associated virus vectors for in vitro and in vivo applications.

Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 1010-1011 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.

A Rare Case of Rush Progression of Purulent Pericarditis by Escherichia coli in a Patient with Malignant Lymphoma.

Purulent pericarditis is a rare disease in the antibiotic era. The common pathogens of purulent pericarditis are gram-positive species such as Staphylococcus aureus. Streptococcus pneumoniae, Salmonella, Haemophilus, fungal pathogens/tuberculosis can also result in purulent pericarditis. We report an old male case of purulent pericarditis by Escherichia coli. He came to our hospital suffering from leg edema for 3 months. Echocardiography revealed the large amount of pericardial effusion, and he was admitted to test the cause of pericardial effusion without high fever, tachycardia, and shock vital signs. On the third day, he suddenly presented vital shock. We performed emergency cardiopulmonary resuscitation and pericardiocentesis. Appearance of pericardial effusion was hemorrhagic and purulent. The gram stain revealed remarkable E. coli invasion to pericardial space. Antibiotic therapy was immediately started; however, he died on sixth day with septic shock. The cytological examination of pericardial effusion suggested the invasion of malignant lymphoma to pericardium. This case showed subacute or chronic process of pericarditis without severe clinical and laboratory sings before admission. Nevertheless, bacterial purulent pericarditis usually shows acute clinical manifestation; the first process of this case was very silent. Immunosuppression of malignant lymphoma might make E. coli translocation from gastrointestinal tract to pericardial space, and bacterial pericarditis was progressed to purulent pericarditis. In the latter process, this case showed unexpected rush progression to death by sepsis from purulent pericarditis. Immediate pericardiocentesis should be performed for a prompt diagnosis of purulent pericarditis, and it might have improved the outcome of this case.

Arterial Stiffness Is Significantly Associated With Left Ventricular Diastolic Dysfunction in Patients With Cardiovascular Disease.

Left ventricular (LV) diastolic dysfunction is considered the main cause of heart failure with preserved ejection fraction (HFpEF). There have been few reports on the correlation between LV diastolic dysfunction and arterial stiffness in patients with clinical cardiovascular disease.This cross-sectional study enrolled 100 patients (67 men, 33 women; mean age, 70 years). All participants were diagnosed with cardiovascular disease. A total of 89 (89%) patients had coronary artery disease or HF. Patients with reduced EF and valvular disease were excluded. Arterial stiffness was assessed by the cardio-ankle vascular index (CAVI), and LV diastolic dysfunction was estimated using echocardiography. The patients were divided into two groups based on the median value of CAVI. In all patients the ratio of early diastolic transmitral flow velocity to early diastolic mitral annular velocity (E/e') was significantly higher in the high CAVI group than in the low CAVI group (15.5 ± 6.4 versus 12.5 ± 2.9, P = 0.003). In the HF subgroup, E/e' was also significantly higher in the high CAVI group than in the low CAVI group (17.2 ± 5.9 versus 13.0 ± 3.1, P = 0.026). In univariate regression analysis, CAVI was significantly associated with E/e' in all patients (ß = 0.28, P = 0.004) and in HF patients (ß = 0.4, P = 0.028). Also in multivariate analysis, CAVI remained as an independent predictive factor of E/e' (ß = 0.252, P = 0.037).A high CAVI was independently associated with LV diastolic dysfunction in patients with clinical cardiovascular disease. These results suggested that arterial stiffness contributed to the development of LV diastolic dysfunction.

Usefulness of the d-ROMs test for prediction of cardiovascular events.

BACKGROUND: d-ROMs test developed to determine the degree of individual oxidative stress may predict cardiovascular events. METHODS AND RESULTS: 265 patients (204 men, 61 women; age, 65±13years) who had been treated for cardiovascular disease were divided evenly by quartile of baseline d-ROMs levels, and were followed up. During the observation periods of 2.66±1.47years, there were 14 (5%) deaths, 8 (3%) cardiovascular deaths, 13 (5%) major adverse cardiovascular events (MACEs), and 51 (19%) all cardiovascular events including heart failure, cardiovascular surgery, and revascularization. Log-rank tests demonstrated that the patients in the 4th quartile (d-ROMs≧395.00U.CARR) had a higher incidence rate of cardiovascular death than those in the 2nd quartile (d-ROMs 286.00-335.00, p=0.022). In multivariate Cox regression analysis, even after adjustment for age, sex, coronary risk factors, C-reactive protein, and renal function, high d-ROMs was a risk factor for all-cause death [adjusted HR of 4th vs. 1st quartile, 10.791 (95% confidence interval 1.032-112.805), p=0.047], and all cardiovascular events [HR of 4th vs. 1st quartile, 2.651 (95% confidence interval 1.138-6.177), p=0.024]. CONCLUSIONS: Our results suggest that d-ROMs is a useful oxidative stress marker to assess prognosis and risk of further cardiovascular events.

Mechanical clot fragmentation using a Swan-Ganz catheter is useful for acute massive pulmonary thromboembolism.

Massive pulmonary thromboembolism (PE) is an acute-onset, life-threatening disease in the perioperative period. Massive PE led to severe hypoxemia and cor pulmonale in our patient who had undergone an operation for thalamic hemorrhage. Mechanical clot fragmentation using a Swan-Ganz catheter was attempted, because thrombolytic therapy was contraindicated for our patient. This procedure was successful, resulting in a decrease in pulmonary artery pressure with improvement in hypoxemia. Thus, this procedure may be useful for patients with massive PE with contraindications to thrombolytic therapy. .

The dramatic 3D IVUS imaging demonstrating a dislodged coronary stent.

A 78-year-old female underwent cardiac evaluation because she had presented with typical effort-induced chest pain. Coronary angiography revealed severe stenoses in the proximal left anterior descending coronary artery (LAD) and ostium of the diagonal branch. Then, a stent was deployed in the LAD covering the takeoff of the branch. Next, we tried to deploy an additional stent in the branch, but the new stent was stripped off the balloon platform. We demonstrate here the dramatic 3D intravascular ultrasound (IVUS) images of a dislodged stent, indicating that 3D IVUS imaging should allow the precise location and statement of lost stent.