Karen Hanley-Yanez

Shared Genetic Predisposition in Peripartum and Dilated Cardiomyopathies

Background Peripartum cardiomyopathy shares some clinical features with idiopathic dilated cardiomyopathy, a disorder caused by mutations in more than 40 genes, including TTN, which encodes the sarcomere protein titin. Methods In 172 women with peripartum cardiomyopathy, we sequenced 43 genes with variants that have been associated with dilated cardiomyopathy. We compared the prevalence of different variant types (nonsense, frameshift, and splicing) in these women with the prevalence of such variants in persons with dilated cardiomyopathy and with population controls. Results We identified 26 distinct, rare truncating variants in eight genes among women with peripartum cardiomyopathy. The prevalence of truncating variants (26 in 172 [15%]) was significantly higher than that in a reference population of 60,706 persons (4.7%, P=1.3×10(-7)) but was similar to that in a cohort of patients with dilated cardiomyopathy (55 of 332 patients [17%], P=0.81). Two thirds of identified truncating variants were in TTN, as seen in 10% of the patients and in 1.4% of the reference population (P=2.7×10(-10)); almost all TTN variants were located in the titin A-band. Seven of the TTN truncating variants were previously reported in patients with idiopathic dilated cardiomyopathy. In a clinically well-characterized cohort of 83 women with peripartum cardiomyopathy, the presence of TTN truncating variants was significantly correlated with a lower ejection fraction at 1-year follow-up (P=0.005). Conclusions The distribution of truncating variants in a large series of women with peripartum cardiomyopathy was remarkably similar to that found in patients with idiopathic dilated cardiomyopathy. TTN truncating variants were the most prevalent genetic predisposition in each disorder.

Evidence for local relaxin ligand-receptor expression and function in arteries

Relaxin is a 6 kDa protein hormone produced by the corpus luteum and secreted into the blood during pregnancy in rodents and humans. Growing evidence indicates that circulating relaxin causes vasodilatation and increases in arterial compliance, which may be among its most important actions during pregnancy. Here we investigated whether there is local expression and function of relaxin and relaxin receptor in arteries of nonpregnant females and males. Relaxin‐1 and its major receptor, Lgr7, mRNA are expressed in thoracic aortas, small renal and mesenteric arteries from mice and rats of both sexes, as well as in small renal arteries from female tammar wallabies (an Australian marsupial). Using available antibodies for rat and mouse Lgr7 receptor and rat relaxin, we also identified protein expression in arteries. Small renal arteries isolated from relaxin‐1 gene‐deficient mice demonstrate enhanced myogenic reactivity and decreased passive compliance relative to wild‐type (WT) and heterozygous mice. Taken together, these findings reveal an arterial‐derived, relaxin ligand‐receptor system that acts locally to regulate arterial function.—Novak, J., Parry, L. J., Matthews, J. E., Kerchner, L. J., Indovina, K., Hanley‐Yanez, K., Doty, K. D., Debrah, D. O., Shroff, S. G., Conrad, K. P. Evidence for local relaxin ligand‐receptor expression and function in arteries. FASEB J. 20, 2352–2362 (2006)

G-protein beta-3 subunit genotype predicts enhanced benefit of fixed-dose isosorbide dinitrate and hydralazine: Results of A-HeFT

OBJECTIVES The purpose of this study was to evaluate the influence of the guanine nucleotide-binding proteins (G-proteins), beta-3 subunit (GNB3) genotype on the effectiveness of a fixed-dose combination of isosorbide dinitrate and hydralazine (FDC I/H) in A-HeFT (African American Heart Failure Trial). BACKGROUND GNB3 plays a role in alpha2-adrenergic signaling. A polymorphism (C825T) exists, and the T allele is linked to enhanced alpha-adrenergic tone and is more prevalent in African Americans. METHODS A total of 350 subjects enrolled in the genetic substudy (GRAHF [Genetic Risk Assessment of Heart Failure in African Americans]) were genotyped for the C825T polymorphism. The impact of FDC I/H on a composite score (CS) that incorporated death, hospital stay for heart failure, and change in quality of life (QoL) and on event-free survival were assessed in GNB3 genotype subsets. RESULTS The GRAHF cohort was 60% male, 25% ischemic, 97% New York Heart Association functional class III, age 57 ± 13 years, with a mean qualifying left ventricular ejection fraction of 0.24 ± 0.06. For GNB3 genotype, 184 subjects were TT (53%), 137 (39%) CT, and 29 (8%) were CC. In GNB3 TT subjects, FDC I/H improved the CS (FDC I/H = 0.50 ± 1.6; placebo = -0.11 ± 1.8, p = 0.02), QoL (FDC I/H = 0.69 ± 1.4; placebo = 0.24 ± 1.5, p = 0.04), and event-free survival (hazard ratio: 0.51, p = 0.047), but not in subjects with the C allele (for CS, FDC I/H = -0.05 ± 1.7; placebo = -0.09 ± 1.7, p = 0.87; for QoL, FDC I/H = 0.28 ± 1.5; placebo = 0.14 ± 1.5, p = 0.56; and for event-free survival, p = 0.35). CONCLUSIONS The GNB3 TT genotype was associated with greater therapeutic effect of FDC I/H in A-HeFT. The role of the GNB3 genotype for targeting therapy with FDC I/H deserves further study.

Detection of decreased cytotoxic effector CD8+ T lymphocytes in atopic dermatitis by flow cytometry

Atopic dermatitis (AD) is a common dermatologic disease of unknown etiology. Patients are admitted with a chronically recurring inflammation of the dermis and epidermis characterized by severe pruritlls.‘. 2 Studies to date have demonstrated immune anomalies associated with AD. A frequently reported observation in patients with AD is a reduction in the peripheral blood of CD8 + T lymphocytes,2 whereas the percentage of CD4+ cells are generally not altered, and altered CD4/CD8 ratios are usually credited to the reduction in CD8 + cells. A previous report by Leung et a1.3 has demonstrated that a decrease in peripheral blood CD8 + lymphocytes is associated with diminished in vitro cytotoxic activity. Unfortunately, the distinction between reduced cytolytic activity caused by decreased numbers of cytotoxic effector cells versus impaired cytolytic abilities of the cells could not be made. Recently a monoclonal antibody (designated S6Fl) has been produced that can distinguish between cytotoxic effector and suppressor effector CD8 + lymphocyte subsets.4 This antibody recognizes a novel epitope of the LFA-1 antigen, which is located on the surface membrane of CD8 + cells. CD8 + , S6Flb”@” cells exhibit cytotoxic activity, whereas CD8 + , S6Fldim lymphocytes manifest suppressor function. In the current study we used flow cytometric immunophenotyping to study lymphocyte subsets, particularly CD8 + subsets defined by the S6Fl antibody and CD4 subsets defined by CD29, in peripheral blood of adult patients with AD. These subsets were examined to better define the immune lesions in AD and

Alterations in cell-surface carbohydrates of rat large granular lymphocytes associated with interleukin-2 activation

The activation of large granular lymphocytes (LGLs)/natural killer (NK) cells with interleukin-2 (IL-2) has been shown to increase the ability of these cells to lyse NK-resistant tumor target cells. Activated LGLs, termed LAK (lymphokine-activated killer) cells, have been demonstrated to be of therapeutic value in vivo against metastatic tumors. The mechanism by which IL-2 induces broadened cytolytic capability, as well as the molecular basis of target recognition and killing by the activated cells has not yet been elucidated. Since carbohydrate moieties have been demonstrated to be of possible significance in the cytolytic cascade of a variety of effector cells, the current study was undertaken to determine if the activation of LGLs with IL-2 is accompanied by an alteration of cell-surface carbohydrates. Two-color flow cytometry was performed to identify LGL/NK cells in populations of nylon wool-nonadherent splenic mononuclear cells and to assess the binding of various lectins to activated as well as nonactivated LGLs. Increases were observed in the binding of four lectins to LGLs after IL-2 activation; Triticum vulgaris (wheat germ agglutinin), Phytolacca americana (pokeweed mitogen), Lycopersicon esculentum (tomato lectin), and Griffonia simplicifolia I-B4 (GSI-B4). The wheat germ, pokeweed, and tomato lectins recognize complex carbohydrates structure consisting of GlcNAc(Bl,4GlcNAc)n while GSI-B4 recognizes alpha-D-galactose terminal end groups. Lectin binding to the activated LGLs was homogenous (i.e., flow cytometry revealed only a single population of fluorescent cells). Lectin binding to LGLs prior to activation was more heterogeneous, however, the tomato lectin uniquely revealed a bimodal distribution of receptors. These data indicate that LGL/NK cells from the rat are heterogeneous in their ability to bind specific lectins, and that IL-2 activation of these cells results in altered expression of specific cell-surface carbohydrates.

GNB3 C825T Polymorphism and Myocardial Recovery in Peripartum Cardiomyopathy Results of the Multicenter Investigations of Pregnancy-Associated Cardiomyopathy Study

Background—Black women are at greater risk for peripartum cardiomyopathy (PPCM). The guanine nucleotide–binding proteins &bgr;-3 subunit (GNB3) has a polymorphism C825T. The GNB3 TT genotype more prevalent in blacks is associated with poorer outcomes. We evaluated GNB3 genotype and myocardial recovery in PPCM. Methods and Results—A total of 97 women with PPCM were enrolled and genotyped for the GNB3 T/C polymorphism. Left ventricular ejection fraction (LVEF) was assessed by echocardiography at entry, 6 and 12 months postpartum. LVEF over time in subjects with the GNB3 TT genotype was compared with those with the C allele overall and in black and white subsets. The cohort was 30% black, age 30+6, LVEF 0.34+0.10 at entry 31+25 days postpartum. The % GNB3 genotype for TT/CT/CC=23/41/36 and differed markedly by race (blacks=52/38/10 versus whites=10/44/46, P<0.001). In subjects with the TT genotype, LVEF at entry was lower (TT=0.31+0.09; CT+CC=0.35+0.09, P=0.054) and this difference increased at 6 (TT=0.45+0.15; CT+CC=0.53+0.08, P=0.002) and 12 months (TT=0.45+0.15; CT+CC=0.56+0.07, P<0.001.). The difference in LVEF at 12 months by genotype was most pronounced in blacks (12 months LVEF for GNB3 TT=0.39+0.16; versus CT+CC=0.53+0.09, P=0.02) but evident in whites (TT=0.50++0.11; CT+CC=0.56+0.06, P=0.04). Conclusions—The GNB3 TT genotype was associated with lower LVEF at 6 and 12 months in women with PPCM, and this was particularly evident in blacks. Racial differences in the prevalence and impact of GNB3 TT may contribute to poorer outcomes in black women with PPCM.Background— Black women are at greater risk for peripartum cardiomyopathy (PPCM). The guanine nucleotide–binding proteins β-3 subunit (GNB3) has a polymorphism C825T. The GNB3 TT genotype more prevalent in blacks is associated with poorer outcomes. We evaluated GNB3 genotype and myocardial recovery in PPCM. Methods and Results— A total of 97 women with PPCM were enrolled and genotyped for the GNB3 T/C polymorphism. Left ventricular ejection fraction (LVEF) was assessed by echocardiography at entry, 6 and 12 months postpartum. LVEF over time in subjects with the GNB3 TT genotype was compared with those with the C allele overall and in black and white subsets. The cohort was 30% black, age 30+6, LVEF 0.34+0.10 at entry 31+25 days postpartum. The % GNB3 genotype for TT/CT/CC=23/41/36 and differed markedly by race (blacks=52/38/10 versus whites=10/44/46, P <0.001). In subjects with the TT genotype, LVEF at entry was lower (TT=0.31+0.09; CT+CC=0.35+0.09, P =0.054) and this difference increased at 6 (TT=0.45+0.15; CT+CC=0.53+0.08, P =0.002) and 12 months (TT=0.45+0.15; CT+CC=0.56+0.07, P <0.001.). The difference in LVEF at 12 months by genotype was most pronounced in blacks (12 months LVEF for GNB3 TT=0.39+0.16; versus CT+CC=0.53+0.09, P =0.02) but evident in whites (TT=0.50++0.11; CT+CC=0.56+0.06, P =0.04). Conclusions— The GNB3 TT genotype was associated with lower LVEF at 6 and 12 months in women with PPCM, and this was particularly evident in blacks. Racial differences in the prevalence and impact of GNB3 TT may contribute to poorer outcomes in black women with PPCM.

Chemokine receptor patterns and right heart failure in mechanical circulatory support

BACKGROUND Right ventricular failure (RVF) complicates 9% to 44% of left ventricular assist device (LVAD) implants post-operatively. Current prediction scores perform only modestly in validation studies, and do not include immune markers. Chemokines are inflammatory signaling molecules with a fundamental role in cardiac physiology and stress adaptation. In this study we investigated chemokine receptor regulation in LVAD recipients who develop RVF. METHODS Expression of chemokine receptor (CCR) genes 3 to 8 were examined in the peripheral blood of 111 LVAD patients, collected 24 hours before implant. RNA was isolated using a PAXgene protocol. Gene expression was assessed using a targeted microarray (RT2 Profiler PCR Array; Qiagen). Results were expressed as polymerase chain reaction (PCR) cycles to threshold and normalized to the average of 3 control genes, glyceraldehyde phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and β2-microglobulin (B2M). Secondary outcomes studied were 1-year mortality and long-term RV failure (RVF-LT). RESULTS CCR3, CCR4, CCR6, CCR7 and CCR8 were downregulated in LVAD recipients with RVF. Within this cohort of patients, CCR4, CCR7 and CCR8 were further downregulated in those who required RV mechanical support. In addition, under-expression of CCR3 to CCR8 was independently associated with an increased risk of mortality at 1 year, even after adjusting for RVF. CCR expression did not predict RVF-LT in our patient cohort. CONCLUSIONS Pre-LVAD CCR downregulation is associated with RVF and increased mortality after implant. Inflammatory signatures may play a major role in prognostication in this patient population.

Association of Variants in BAG3 With Cardiomyopathy Outcomes in African American Individuals

Importance The prevalence of nonischemic dilated cardiomyopathy (DCM) is greater in individuals of African ancestry than in individuals of European ancestry. However, little is known about whether the difference in prevalence or outcomes is associated with functional genetic variants. Objective We hypothesized that Bcl2-associated anthanogene 3 (BAG3) genetic variants were associated with outcomes in individuals of African ancestry with DCM. Design This multicohort study of the BAG3 genotype in patients of African ancestry with dilated cardiomyopathy uses DNA obtained from African American individuals enrolled in 3 clinical studies: the Genetic Risk Assessment of African Americans With Heart Failure (GRAHF) study; the Intervention in Myocarditis and Acute Cardiomyopathy Trial-2 (IMAC-2) study; and the Genetic Risk Assessment of Cardiac Events (GRACE) study. Samples of DNA were also acquired from the left ventricular myocardium of patients of African ancestry who underwent heart transplant at the University of Colorado and University of Pittsburgh. Main Outcomes and Measures The primary end points were the prevalence of BAG3 mutations in African American individuals and event-free survival in participants harboring functional BAG3 mutations. Results Four BAG3 genetic variants were identified; these were expressed in 42 of 402 African American individuals (10.4%) with nonischemic heart failure and 9 of 107 African American individuals (8.4%) with ischemic heart failure but were not present in a reference population of European ancestry (P < .001). The variants included 2 nonsynonymous single-nucleotide variants; 1 three-nucleotide in-frame insertion; and 2 single-nucleotide variants that were linked in cis. The presence of BAG3 variants was associated with a nearly 2-fold (hazard ratio, 1.97 [95% CI, 1.19-3.24]; P = .01) increase in cardiac events in carriers compared with noncarriers. Transfection of transformed adult human ventricular myocytes with plasmids expressing the 4 variants demonstrated that each variant caused an increase in apoptosis and a decrease in autophagy when samples were subjected to the stress of hypoxia-reoxygenation. Conclusions and Relevance This study demonstrates that genetic variants in BAG3 found almost exclusively in individuals of African ancestry were not causative of disease but were associated with a negative outcome in patients with a dilated cardiomyopathy through modulation of the function of BAG3. The results emphasize the importance of biological differences in causing phenotypic variance across diverse patient populations, the need to include diverse populations in genetic cohorts, and the importance of determining the pathogenicity of genetic variants.

Maternal Obesity Affects Cardiac Remodeling and Recovery in Women with Peripartum Cardiomyopathy

Objective To examine the association between maternal obesity on left ventricular (LV) size and recovery in women with peripartum cardiomyopathy (PPCM). Study Design This was a prospective analysis of 100 women enrolled within 13 weeks of PPCM diagnosis and followed for a year in the Investigation of Pregnancy Associated Cardiomyopathy study. Adiposity was defined by standard body mass index (BMI) definitions for under/normal weight, overweight, and obesity. Demographic, clinical, and biomarker variables were compared across weight categories. Outcomes LV end‐diastolic diameter (LVEDD) and ejection fraction were measured at entry, 6, and 12 months postpartum. Multivariable regression models examined the relationship between adiposity, LV size, and leptin levels with cardiac recovery at 6 and 12 months postpartum. Results Obese and nonobese women had similar LV dysfunction at entry. Obese women had greater LV size and less LV recovery at 6 and 12 months postpartum. BMI was positively associated with leptin and ventricular diameter. Greater BMI at entry remained associated with less ventricular recovery at 6 months (p = 0.02) in adjusted race‐stratified models. LVEDD at entry predicted lower ejection fraction at 6 months (p < 0.001) and similarly at 12 months. Conclusion Obese women with PPCM had greater cardiac remodeling, higher leptin levels, and diminished cardiac recovery.

G-PROTEIN RECEPTOR KINASE 5 POLYMORPHISMS AND OUTCOMES IN THE AFRICAN AMERICAN HEART FAILURE TRIAL: RESULTS FROM THE GENETIC RISK ASSESSMENT OF HEART FAILURE IN AFRICAN-AMERICANS SUB-STUDY

Background: G-protein receptor kinases (GRKs) are central to beta receptor inactivation. A polymorphism for GRK5, common in African Americans, substitutes leucine (T allele) for glutamine (A allele) at amino acid 41. GRK5 Leu41 boosts beta receptor desensitization, and may impact heart failure