Chuen-Den Tseng

Angiotensin II Activates Signal Transducer and Activators of Transcription 3 via Rac1 in Atrial Myocytes and Fibroblasts Implication for the Therapeutic Effect of Statin in Atrial Structural Remodeling

Background— Recently, activation of the local renin-angiotensin system and mitogen-activated protein kinase pathways in atrial myocardium has been found to play an important role in atrial structural remodeling related to atrial fibrillation. Another important mediator of the angiotensin II (Ang II) effect is the Janus kinase/signal transducers and activators of transcription (STAT) pathway, which has never been characterized in the atrium. Methods and Results— In cultured atrial myocytes and fibroblasts, Ang II induced tyrosine phosphorylation of STAT3 through a Rac1-dependent mechanism, which was inhibited by dominant-negative Rac1, losartan, and simvastatin. In atrial myocytes, activation of STAT3 by Rac1 was mediated by direct association of Rac1 with STAT3; however, in atrial fibroblasts, it was mediated by an indirect paracrine effect. Constitutively active STAT3 increased protein synthesis, and dominant-negative STAT3 abrogated Ang II-induced protein synthesis in atrial myocytes and fibroblasts. Rats infused long term with Ang II exhibited higher levels of activated Rac1, phospho-STAT3, collagen synthesis, and atrial fibrosis in the atria, all of which were attenuated by oral losartan and simvastatin. In human atrial tissues from patients with atrial fibrillation, Ang II and phospho-STAT3 levels were also elevated. Conclusions— The Ang II/Rac1/STAT3 pathway is an important signaling pathway in the atrial myocardium to mediate atrial structural remodeling, and losartan and statin may be able to reverse Ang II-induced atrial structural remodeling in atrial fibrillation.

Serum bilirubin is inversely associated with insulin resistance and metabolic syndrome among children and adolescents.

OBJECTIVE Bilirubin is a potent antioxidant and a cyroprotectant. Low serum bilirubin is associated with atherosclerosis. Little is known about its role in metabolic syndrome (MS) among children and adolescents. METHODS We examined 4723 children and adolescents aged 12-17 years with reliable measures of various serum hepatic profiles and metabolic risks from Health and Nutrition Examination Survey 1999-2004. RESULTS The results showed that the prevalence of the MS was from 6.6+/-1.2% in the lowest quartile to 2.1+/-1.9% in the highest quartile of the concentration of total bilirubin. The graded association remained significant after the adjustment of other co-variates. The odds ratios for the MS were around 0.29 (0.08-0.99) and 0.23 (0.08-0.65) for the upper two quartiles when using the lowest quartile as reference for the concentration of total bilirubin. The quartiles of the serum total bilirubin levels were inversely correlated with the homeostasis model assessment (HOMA-IR) and insulin while not associated with the serum C-reactive protein (CRP) levels. CONCLUSIONS The serum total bilirubin levels are inversely correlated with the prevalence of the MS. The mechanism of the association between MS and total bilirubin may be related to the insulin resistance status.

Increased Expression of Mineralocorticoid Receptor in Human Atrial Fibrillation and a Cellular Model of Atrial Fibrillation

OBJECTIVES This study was designed to evaluate the status of steroidogenesis proteins and de novo synthesis of aldosterone in the atrium, and relationships of these factors to atrial fibrillation (AF). BACKGROUND The role of mineralocorticoid in the pathogenesis of AF is unknown. METHODS We studied atrial expression of steroidogenesis proteins and aldosterone level in patients with and without AF, and in HL-1 atrial myocytes. We also investigated the electrophysiologic effects and signal transduction of aldosterone on atrial myocytes. RESULTS We found basal expressions of mineralocorticoid receptors (MRs), glucocorticoid receptors, and 11-beta-hydroxysteroid dehydrogenase type 2 (11bHSD2) but not 11-beta-hydroxylase (CYP11B1) or aldosterone synthase (CYP11B2) in human atria and HL-1 myocytes. There was no significant difference of mean atrial aldosterone level between patients with AF and those with normal sinus rhythm. However, patients with AF had a significantly higher atrial MR expression compared with those with normal sinus rhythm (1.73 +/- 0.24-fold, p < 0.001). Using mouse HL-1 atrial myocytes as a cellular AF model, we found that rapid depolarization increased MR expression (1.97 +/- 0.72-fold, p = 0.008) through a calcium-dependent mechanism, thus augmenting the genomic effect of aldosterone signaling as evaluated by MR reporter. Aldosterone increased intracellular oxidative stress through a nongenomic pathway, which was attenuated by nicotinamide adenine dinucleotide phosphate oxidase inhibitor diphenyleneiodonium, but not by MR-blockade spironolactone. Aldosterone increased expression of the alpha-1G and -1H subunits of the T-type calcium channel and thus increased the T-type calcium current (-13.6 +/- 2.9 pA/pF vs. -4.5 +/- 1.6 pA/pF, p < 0.01) and the intracellular calcium load through a genomic pathway, which were attenuated by spironolactone, but not by diphenyleneiodonium. CONCLUSIONS Expression of MR increased in AF, thus augmenting the genomic effects of aldosterone. Aldosterone induced atrial ionic remodeling and calcium overload through a genomic pathway, which was attenuated by spironolactone. These results suggest that aldosterone may play a role in AF electrical remodeling and provide insight into the treatment of AF with MR blockade.

Angiotensin II Increases Expression of α1C Subunit of L-Type Calcium Channel Through a Reactive Oxygen Species and cAMP Response Element–Binding Protein–Dependent Pathway in HL-1 Myocytes

Angiotensin II (Ang II) is involved in the pathogenesis of atrial fibrillation (AF). L-type calcium channel (LCC) expression is altered in AF remodeling. We investigated whether Ang II modulates LCC current through transcriptional regulation, by using murine atrial HL-1 cells, which have a spontaneous calcium transient, and an in vivo rat model. Ang II increased LCC α1C subunit mRNA and protein levels and LCC current density, which resulted in an augmented calcium transient in atrial myocytes. An ≈2-kb promoter region of LCC α1C subunit gene was cloned to the pGL3 luciferase vector. Ang II significantly increased promoter activity in a concentration- and time-dependent manner. Truncation and mutational analysis of the LCC α1C subunit gene promoter showed that cAMP response element (CRE) (−1853 to −1845) was an important cis element in Ang II-induced LCC α1C subunit gene expression. Transfection of dominant-negative CRE binding protein (CREB) (pCMV-CREBS133A) abolished the Ang II effect. Ang II (1 μmol/L, 2 hours) induced serine 133 phosphorylation of CREB and binding of CREB to CRE and increased LCC α1C subunit gene promoter activity through a protein kinase C/NADPH oxidase/reactive oxygen species pathway, which was blocked by the Ang II type 1 receptor blocker losartan and the antioxidant simvastatin. In the rat model, Ang II infusion increased LCC α1C subunit expression and serine 133 phosphorylation of CREB, which were attenuated by oral losartan and simvastatin. In summary, Ang II induced LCC α1C subunit expression via a protein kinase C–, reactive oxygen species–, and CREB-dependent pathway and was blocked by losartan and simvastatin.

Molecular variant M235T of the angiotensinogen gene is associated with essential hypertension in Taiwanese

Objective To examine the association of the molecular variants of the angiotensinogen (AGT) gene with essential hypertension in Taiwanese. Methods We conducted a case-control study concerning 151 subjects, 102 hypertensives and 49 normotensives. We created a rapid mini-sequencing method based on dye-terminator cycle sequencing to simultaneously detect the M235T and T174M variants of the AGT gene for each subject. Results The genotype and allele distribution of the M235T variant differed significantly in hypertensives and normotensives (χ2 = 11.106, P = 0.004 and χ2 = 6.453, P = 0.011, respectively), whereas those of the T174M variant did not differ (χ2 = 0.004, P = 0.998 and χ2 = 0.032, P = 0.858, respectively). The odds ratio for hypertension was 3.64 (95% confidence interval 1.56–8.49) for subjects with the C/C genotype of the M235T variant compared with other genotypes or 2.87 (95% confidence interval 1.76–4.68) for those carrying allele C versus those carrying allele T. Conclusion The molecular variant M235T, but not T174M, of the AGT gene is associated significantly with essential hypertension in this Taiwanese population. The genotype C/C or allele C is a risk factor for hypertension. The underlying mechanism of this association needs to be elucidated further.

Plasma levels of tumor necrosis factor-α and interleukin-6 are associated with diastolic heart failure through downregulation of sarcoplasmic reticulum Ca2+ ATPase.

Objective:The inflammatory process is associated with cardiac diastolic dysfunction, which has been demonstrated to be an independent prognostic marker for the mortality of critically ill patients. We investigated the association among inflammatory cytokines (tumor necrosis factor-&agr; and interleukin-6), diastolic heart failure, and the possible molecular mechanism. Design:Prospective case-controlled cohort and molecular studies. Setting:University hospital and research laboratory. Subjects:Patients with a diagnosis of diastolic heart failure by echocardiography and matched control subjects from the general population (study group 1) and also subjects from the intensive care unit (study group 2). Sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene expression and diastolic calcium decay in HL-1 cardiomyocytes were used as molecular phenotypes of diastolic heart failure. Interventions:Soluble plasma levels of tumor necrosis factor-&agr; and interleukin-6 were measured in all subjects. An approximate 1.75-kb promoter of the SERCA2 gene was cloned to the pGL3 luciferase reporter. The effect of tumor necrosis factor-&agr; and interleukin-6 on SERCA2 gene expression and diastolic calcium decay of HL-1 cardiomyocytes were investigated. Measurements and Main Results:Patients with diastolic heart failure had significantly higher plasma levels of tumor necrosis factor-&agr; and interleukin-6 than the control subjects. Significant correlations (p < .01 for each) were found for tumor necrosis factor-&agr; and E/Em (r = .87) and E/A (r = −0.69), and for interleukin-6 and E/Em (r = .80) and E/A (r = −0.65). Cytokine levels were also correlated with diastolic function in critically ill patients (study group 2), and diastolic function improved significantly in association with decrease of cytokines. Tumor necrosis factor-&agr;, interleukin-6, and sera from critically ill patients downregulated the expression of the SERCA2 gene. Tumor necrosis factor-&agr; and interleukin-6 also delayed the diastolic calcium reuptake and decay in cardiomyocytes. Conclusions:Through downregulation of SERCA2 gene expression, inflammatory cytokines may cause cardiac diastolic dysfunction by decreasing diastolic calcium reuptake. Our study may suggest novel therapeutic strategies for diastolic heart failure and critically ill patients by modulating inflammatory reactions.

Lack of association of the angiotensin converting enzyme gene polymorphism with essential hypertension in a chinese population

To examine the association between insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene and essential hypertension in a Chinese population, a case-control study was conducted using 157 hypertensive and 115 normotensive subjects. The I/D polymorphism of the ACE gene was identified by polymerase chain reaction. Plasma ACE activity was determined using spectrophotometry. The difference of allele frequencies between normotensives and hypertensives was statistically significant (chi 2 = 4.467, P = .035), while the genotype distribution was not different between normotensive and hypertensive subjects (chi 2 = 3.954, P = .138). Plasma ACE activity was highest in the DD genotype, followed by the ID genotype, and the lowest in the II genotype (P = .0001 in normotensives and P = .163 in hypertensives, respectively). Thus, we conclude that the ACE gene polymorphism is not associated with essential hypertension in this Chinese population, but plasma ACE activity is genetically determined in the normotensive Chinese.

Renin-angiotensin system component expression in the HL-1 atrial cell line and in a pig model of atrial fibrillation.

Objectives Local atrial tissue angiotensin II (AngII) level is elevated in atrial fibrillation (AF), but the mechanism is unknown. We hypothesized that atrial myocytes express all components of the renin–angiotensin system (RAS) and investigated whether rapid depolarization alone is sufficient to increase paracrine AngII production by up-regulating RAS component expression. Methods In the HL-1 atrial cell line, rapid depolarization was induced by rapid field electrical stimulation (RES) at 1.0 V/cm and 600/min (10 Hz) in atrial HL-1 cells. In a pig model of AF, AF was induced by atrial pacing at 600/min in 10 adult pigs and 10 sham-operated pigs for comparison. Results In atrial myocytes, RES induced a sustained elevation of intracellular calcium, and up-regulation of angiotensin-converting enzyme (ACE), chymase and angiotensinogen, resulting in increased AngII production. RES-induced AngII production was attenuated by enalapril [ACE inhibitor (ACEI)] and chymostatin (chymase inhibitor). Conditioned medium from RES-stimulated atrial myocytes increased [3H]leucine uptake and atrial natriuretic peptide expression in atrial myocytes, and [3H]proline uptake and collagen type 1 alpha 1 expression in atrial fibroblasts. Both were attenuated by co-incubation with the AngII type 1 receptor blocker (ARB) losartan. In the porcine model, significant structural changes and a similar pattern of changes of RAS components were noted in AF pigs. Conclusions Atrial cells expressed all components of RAS and rapid depolarization alone was sufficient to up-regulate RAS components, increase paracrine AngII production and induce atrial structural changes, which are attenuated by ACEI, ARB and chymase inhibitor.

Mechanical Stretch of Atrial Myocyte Monolayer Decreases Sarcoplasmic Reticulum Calcium Adenosine Triphosphatase Expression and Increases Susceptibility to Repolarization Alternans

OBJECTIVES The purpose of this study was to investigate the effect of stretch (the major risk factor for atrial fibrillation [AF]) on spatial and temporal alternations of action potential duration (APD-ALT) and calcium transient in cultured atrial myocyte monolayer. BACKGROUND How rapid firings or premature beats trigger AF is not completely understood. Discordant repolarization alternans, characterized by simultaneous prolongation and shortening of APD in different myocardial regions, is central to the genesis of ventricular fibrillation. We hypothesized that repolarization alternans also is central to the initiation of multiple re-entry circuits and AF. METHODS Confluent HL-1 atrial myocyte monolayer with spontaneous depolarization was cultured in silicone membrane and subjected to mechanical stretch. Rapid field pacing was used to induce alternans. A high-resolution dual optical mapping system was used to record action potentials and calcium transients. RESULTS High-rate pacing induced APD-ALT and calcium transient in atrial myocyte monolayer. Mechanical stretch significantly decreased the thresholds for APD-ALT and calcium transient. Mechanical stretch decreased the expression of sarcoplasmic reticulum adenosine triphosphatase 2, and thus slower calcium reuptake kinetics, which was responsible for the susceptibility to alternans. Mechanical stretch did not alter the APD restitution kinetics. Mechanical stretch also enhanced spatially discordant alternans. Overexpression of sarcoplasmic reticulum adenosine triphosphatase 2 reversed all the effects of stretch on susceptibility to alternans. In intact atrium, mechanical stretch also enhanced discordant alternans. CONCLUSIONS Mechanical stretch increased the susceptibility to alternans in atrial myocytes, which may explain the susceptibility to AF in conditions of atrial stretch, such as mitral valvular heart disease, heart failure, and hypertension.

In-vitro recording of adult zebrafish heart electrocardiogram — A platform for pharmacological testing

BACKGROUND Recently zebrafish has become a powerful vertebrate model system for cardiac arrhythmia and another advantage is that the heart could be rapidly excised for in-vitro electrophysiological recording. METHODS We made direct in-vitro recordings of adult zebrafish heart ECG using the microelectrode and tested the effects of various drugs on zebrafish heart ECG. RESULTS The QT interval change in the ECG was verified by optically mapped action potential duration (APD). The mean in-vitro heart rate (HR) of the adult zebrafish was 118 ± 22 beats/min. The mean QT interval was 312 ± 36 ms and mean HR corrected QT interval (QTc) 439 ± 39 ms. The mean optically mapped APD was 308 ± 30 ms, which was close to the QT interval in ECG (p=0.815). We found that sympathomimetic drug isoproterenol increased HR, whereas L-type calcium channel blocker verapamil decreased HR. Sodium channel blocker quinidine and L-type calcium channel activator BayK8644 prolonged QTc interval in a dose-dependent manner (515 ± 24 ms and 519 ± 27 ms, respectively, both p<0.01). The APD was also prolonged accordingly. Both rapidly and slowly activating delayed rectifier potassium channel (IKr and IKs) blockers E4031 and chromanol 293B, respectively, also prolonged QTc interval in a dose-dependent manner (523 ± 25 ms and 529 ± 27 ms, respectively, both p<0.01). Quinidine, E4031 and chromanol 293B also decreased HR. CONCLUSIONS Direct in-vitro recording of adult zebrafish heart is an efficient platform for test of drugs which exert electrophysiological effects on cardiomyocytes, and perturbation of ionic channels that are responsible for human long QT syndrome also modulates QT interval in adult zebrafish heart.