Myeong-Chan Cho

Transgenic Mice with Cardiac Overexpression of α1B-Adrenergic Receptors IN VIVO α1-ADRENERGIC RECEPTOR-MEDIATED REGULATION of β-ADRENERGIC SIGNALING

Transgenic mice were generated with cardiac-specific overexpression of the wild-type (WT) α1B-adrenergic receptor (AR) using the murine α-myosin heavy chain gene promoter. Previously, we described transgenic mice with α-myosin heavy chain-directed expression of a constitutively active mutant α1B-AR that had a phenotype of myocardial hypertrophy (Milano, C. A., Dolber, P. C., Rockman, H. A., Bond, R. A., Venable M. E., Allen, L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10109–10113). In animals with >40-fold WT α1-AR overexpression, basal myocardial diacylglycerol content was significantly increased, indicating enhanced α1-adrenergic signaling and phospholipase C activity. In contrast to the mice overexpressing constitutively active mutant α1B-ARs, the hearts of these mice did not develop cardiac hypertrophy despite an 8-fold increase in ventricular mRNA for atrial natriuretic factor. In vivo physiology was studied in anesthetized intact animals and showed left ventricular contractility in response to the β-agonist isoproterenol to be significantly depressed in animals overexpressing WT α1B-ARs. Membranes purified from the hearts of WT α1BAR-overexpressing mice demonstrated significantly attenuated adenylyl cyclase activity basally and after stimulation with isoproterenol, norepinephrine, or phenylephrine. Interestingly, thesein vitro changes in signaling were reversed after treating the mice with pertussis toxin, suggesting that the extraordinarily high levels of WT α1B-ARs can lead to coupling to pertussis toxin-sensitive G proteins. Another potential contributor to the observed decreased myocardial signaling and function could be enhanced β-AR desensitization as β-adrenergic receptor kinase (βARK1) activity was found to be significantly elevated (>3-fold) in myocardial extracts isolated from WT α1B-AR-overexpressing mice. This type of altered signal transduction may become critical in disease conditions such as heart failure where βARK1 levels are elevated and β-ARs are down-regulated, leading to a higher percentage of cardiac α1-ARs. Thus, these mice serve as a unique experimental model to study the in vivo interactions between α- and β-ARs in the heart.

Defective beta-adrenergic receptor signaling precedes the development of dilated cardiomyopathy in transgenic mice with calsequestrin overexpression.

Calsequestrin is a high capacity Ca2+-binding protein in the junctional sarcoplasmic reticulum that forms a quaternary complex with junctin, triadin, and the ryanodine receptor. Transgenic mice with cardiac-targeted calsequestrin overexpression show marked suppression of Ca2+-induced Ca2+ release, myocyte hypertrophy, and premature death by 16 weeks of age (Jones, L. R., Suzuki, Y. J., Wang, W., Kobayashi, Y. M., Ramesh, V., Franzini-Armstrong, C., Cleemann, L., and Morad, M. (1998)J. Clin. Invest. 101, 1385–1393). To investigate whether alterations in intracellular Ca2+ trigger changes in the β-adrenergic receptor pathway, we studied calsequestrin overexpressing transgenic mice at 7 and 14 weeks of age. As assessed by echocardiography, calsequestrin mice at 7 weeks showed mild left ventricular enlargement, mild decreased fractional shortening with increased wall thickness. By 14 weeks, the phenotype progressed to marked left ventricular enlargement and severely depressed systolic function. Cardiac catheterization in calsequestrin mice revealed markedly impaired β-adrenergic receptor responsiveness in both 7- and 14- week mice. Biochemical analysis in 7- and 14-week mice showed a significant decrease in total β-adrenergic receptor density, adenylyl cyclase activity, and the percent high affinity agonist binding, which was associated with increased β-adrenergic receptor kinase 1 levels. Taken together, these data indicate that alterations in β-adrenergic receptor signaling precede the development of overt heart failure in this mouse model of progressive cardiomyopathy.

A genome-wide association study of a coronary artery disease risk variant

Although over 30 common genetic susceptibility loci have been identified to be independently associated with coronary artery disease (CAD) risk through genome-wide association studies (GWAS), genetic risk variants reported to date explain only a small fraction of heritability. To identify novel susceptibility variants for CAD and confirm those previously identified in European population, GWAS and a replication study were performed in the Koreans and Japanese. In the discovery stage, we genotyped 2123 cases and 3591 controls with 521 786 SNPs using the Affymetrix SNP Array 6.0 chips in Korean. In the replication, direct genotyping was performed using 3052 cases and 4976 controls from the KItaNagoya Genome study of Japan with 14 selected SNPs. To maximize the coverage of the genome, imputation was performed based on 1000 Genome JPT+CHB and 5.1 million SNPs were retained. CAD association was replicated for three GWAS-identified loci (1p13.3/SORT1 (rs599839), 9p21.3/CDKN2A/2B (rs4977574), and 11q22.3/ PDGFD (rs974819)) in Koreans. From GWAS and a replication, SNP rs3782889 showed a strong association (combined P=3.95 × 10−14), although the association of SNP rs3782889 doesn’t remain statistically significant after adjusting for SNP rs11066015 (proxy SNP with BRAP (r2=1)). But new possible CAD-associated variant was observed for rs9508025 (FLT1), even though its statistical significance did marginally reach at the genome-wide a significance level (combined P=6.07 × 10−7). This study shows that three CAD susceptibility loci, which were previously identified in European can be directly replicated in Koreans and also provides additional evidences implicating suggestive loci as risk variants for CAD in East Asian.

Cardiac remodeling and atrial fibrillation in transgenic mice overexpressing junctin

Junctin is a 26‐kDa integral membrane protein, colocalized with the ryanodine receptor (RyR) and calsequestrin at the junctional sarcoplasmic reticulum (SR) membrane in cardiac and skeletal muscles. To elucidate the functional role of junctin in heart, transgenic (TG) mice overexpressing canine junctin (24–29 folds) under the control of mouse α‐myosin heavy chain promoter were generated. Overexpression of the junctin in mouse heart was associated with heart enlargements, bradycardia, atrial fibrillation, and increased fibrosis. Many ultrastructural alterations were observed in TG atria. The junctional SR cisternae facing transverse‐tubules contained a dense matrix of calsequestrin in TG heart. According to echocardiography, TG mice showed enlarged left ventricles, dilated right atriums, and ventricles with paradoxical septal motion and impaired left ventricular systolic function. Overexpression of junctin led to down‐regulation of triadin and RyR but to up‐regulation of dihydropyridine receptor. The L‐type Ca2+ current density and action potential durations increased, which could be the cause for the bradycardia in TG heart. This study provides an important example of pathogenesis leading to substantial cardiac remodeling and atrial fibrillation, which was caused by overexpression of junctin in heart.

Effects of bone marrow derived mesenchymal stem cells transplantation in acutely infarcting myocardium

Cellular cardiomyoplasty (CCM) is considered to be a novel therapeutic approach for post‐myocardial infarction (MI) heart failure. In this study, the functional effects of cultured mesenchymal stem cells (MSCs) transplantation and the associated histopathologic changes were evaluated in a rat model of MI.

Enhanced Contractility and Decreased β-Adrenergic Receptor Kinase-1 in Mice Lacking Endogenous Norepinephrine and Epinephrine

BACKGROUND Elevated circulating norepinephrine (NE) has been implicated in causing the profound beta-adrenergic receptor (betaAR) downregulation and receptor uncoupling that are characteristic of end-stage human dilated cardiomyopathy, a process mediated in part by increased levels of beta-adrenergic receptor kinase (betaARK1). To explore whether chronic sustained NE stimulation is a primary stimulus that promotes deterioration in cardiac signaling, we characterized a gene-targeted mouse in which activation of the sympathetic nervous system cannot lead to an elevation in plasma NE and epinephrine. METHODS AND RESULTS Gene-targeted mice that lack dopamine beta-hydroxylase (dbh-/-), the enzyme needed to convert dopamine to NE, were created by homologous recombination. In vivo contractile response to the beta1AR agonist dobutamine, measured by a high-fidelity left ventricular micromanometer, was enhanced in mice lacking the dbh gene. In unloaded adult myocytes isolated from dbh-/- mice, basal contractility was significantly increased compared with control cells. Furthermore, the increase in betaAR responsiveness and enhanced cellular contractility were associated with a significant reduction in activity and protein level of betaARK1 and increased high-affinity agonist binding without changes in betaAR density or G-protein levels. CONCLUSIONS Mice that lack the ability to generate NE or epinephrine show increased contractility associated primarily with a decrease in the level of betaARK1 protein and kinase activity. This animal model will be valuable in testing whether NE is required for the pathogenesis of heart failure through mating strategies that cross the dbh-/- mouse into genetically engineered models of heart failure.

Novel Oral Formulation of Paclitaxel Inhibits Neointimal Hyperplasia in a Rat Carotid Artery Injury Model

Background—Paclitaxel has been shown to inhibit vascular smooth muscle cell migration and proliferation contributing to neointimal formation. This study tested whether novel oral formulations of paclitaxel can prevent neointimal formation in a rat carotid artery injury model. Methods and Results—Oral formulations of paclitaxel (0, 5, 7.5, or 10 mg/kg) were administered to 40 rats by gavage for 5 days after injury. The peak plasma levels of paclitaxel administered at 5, 7.5, and 10 mg/kg were 61±16, 89±22, and 108±28 nmol/L, respectively. Treatment effects were assessed 11 days after injury. The angiographic minimum luminal diameters of the oral paclitaxel groups treated at 5, 7.5, and 10 mg/kg were 6.28±2.09, 6.97±1.79, and 7.97±1.57 AU, and these were significantly larger than that of the control group (4.67±1.45 AU). The oral paclitaxel groups (5, 7.5, 10 mg/kg; 0.05±0.05, 0.04±0.03, 0.05±0.03 mm2) showed significant neointimal formation reductions versus the control group (0.13±0.05 mm2). All rats survived to study completion. Only 2 animals in the 10 mg/kg group experienced weight loss (≈10%) and loose stools between 4 and 6 days after injury. All other animals appeared healthy during the study. For comparison purposes, intraperitoneal formulations of paclitaxel (0 or 2 mg/kg) were administered by injection to 15 rats. We confirmed that the intraperitoneal administration of paclitaxel also effectively inhibited neointimal formation. Conclusions—Oral formulations of paclitaxel provide an effective means of inhibiting proliferative response to vascular injury in the rat. Thus, oral formulations of paclitaxel may prevent human restenosis without significant toxicity.

Expression of human Calbindin-D9k correlated with age, vitamin D receptor and blood calcium level in the gastrointestinal tissues

OBJECTIVE Calbindin-D(9k) (CaBP-9k) has been thought to be one of the important factors of calcium absorption and metabolism in the intestine. The understanding of CaBP-9k role in the gastrointestinal tissues is important in human in relation with aging and gender, and whether there is any relationship with mineral homeostasis and risk for the development of age-related calcium disorders such as osteoporosis. DESIGN AND METHODS The expression of CaBP-9k and vitamin D receptor (VDR) was analyzed in the human gastrointestinal tissues with different ages and gender. In addition, the correlation among CaBP-9k expression, age and calcium concentration in blood was elucidated in these tissues. RESULTS A significant increase in the expression of CaBP-9k mRNA was observed in the bulb and 2(nd) portion of duodenum over age in both men and women. However, no significant age-associated change in VDR mRNA level was detected in the duodenum. The concentration of blood calcium level decreased with age in both men and women. CONCLUSION The duodenal CaBP-9k may be not involved in calcium absorption in these tissues. The functional significance of the increase in the intestinal CaBP-9k expression with age is not currently clear and requires further investigation.

Ubiquitous Polygenicity of Human Complex Traits: Genome-Wide Analysis of 49 Traits in Koreans

Recent studies in population of European ancestry have shown that 30%∼50% of heritability for human complex traits such as height and body mass index, and common diseases such as schizophrenia and rheumatoid arthritis, can be captured by common SNPs and that genetic variation attributed to chromosomes are in proportion to their length. Using genome-wide estimation and partitioning approaches, we analysed 49 human quantitative traits, many of which are relevant to human diseases, in 7,170 unrelated Korean individuals genotyped on 326,262 SNPs. For 43 of the 49 traits, we estimated a nominally significant (P<0.05) proportion of variance explained by all SNPs on the Affymetrix 5.0 genotyping array (). On average across 47 of the 49 traits for which the estimate of is non-zero, common SNPs explain approximately one-third (range of 7.8% to 76.8%) of narrow sense heritability. The estimate of is highly correlated with the proportion of SNPs with association P<0.031 (r 2 = 0.92). Longer genomic segments tend to explain more phenotypic variation, with a correlation of 0.78 between the estimate of variance explained by individual chromosomes and their physical length, and 1% of the genome explains approximately 1% of the genetic variance. Despite the fact that there are a few SNPs with large effects for some traits, these results suggest that polygenicity is ubiquitous for most human complex traits and that a substantial proportion of the “missing heritability” is captured by common SNPs.

Impact of Body Mass Index and Waist-to-Hip Ratio on Clinical Outcomes in Patients With ST-Segment Elevation Acute Myocardial Infarction (from the Korean Acute Myocardial Infarction Registry)

The relation between body mass index (BMI) and waist-to-hip ratio (WHR) to clinical outcomes in patients with ST-segment elevation acute myocardial infarction (MI) has not been well described. As part of the Korean Acute MI Registry, we enrolled 3,734 eligible patients who were diagnosed with ST-segment elevation acute MI. The study population was categorized by BMI (into 4 groups according to the World Health Organization classification for the Asian population) and WHR (into 2 sets of 4 groups, 1 set for men and another for women, based on the INTERHEART study). Baseline characteristics and clinical outcomes were analyzed and compared among the BMI and WHR categories. Mean follow-up duration was 199 +/- 37 days. In the BMI category, underweight versus obese patients were older, were more likely to present with heart failure, and underwent guideline-based treatments less frequently. In the WHR category, the reverse trends were apparent for the latter factors except treatment-use frequencies. The highest mortality rate was observed in patients with the lowest BMI and the highest WHR. In an adjusted model, the highest WHR (hazard ratio 5.57, 95% confidence interval 1.53 to 12.29, p = 0.009) and the underweight (hazard ratio 2.88, 95% confidence interval 1.17 to 6.08, p = 0.021) categories within the 2 anthropometric indexes remained as mortality risk factors. In conclusion, the relation between obesity and prognosis after ST-segment elevation acute MI appears complex and should be further assessed in larger population-based cohort studies to determine the associations apparent in this study.