Redfield, Margaret M.

Background— Reductions in numbers of circulating progenitor cells (CD34+ cell subsets) have been demonstrated in patients at risk for, or in the presence of, cardiovascular disease. The mediators of these reductions remain undefined. To determine whether neurohumoral factors might regulate circulating CD34+ cell subsets in vivo, we studied complementary canine models of left ventricular (LV) dysfunction. Methods and Results— A pacing model of severe LV dysfunction and a hypertensive renal wrap model in which dogs were randomized to receive deoxycorticosterone acetate (DOCA) were studied. Circulating CD34+ cell subsets including hematopoietic precursor cells (HPCs: CD34+/CD45 dim /VEGFR2−) and endothelial progenitor cells (EPCs: CD34+/CD45−/VEGFR2+) were quantified. Additionally, the effect of mineralocorticoid excess on circulating progenitor cells in normal dogs was studied. The majority of circulating CD34+ cells expressed CD45dimly and did not express VEGFR2, consistent with an HPC phenotype. HPCs were decreased in response to pacing, and this decrease correlated with plasma aldosterone levels (Spearman rank correlation=−0.67, P =0.03). In the hypertensive renal wrap model, administration of DOCA resulted in decreased HPCs. No changes were seen in EPCs in either model. Normal dogs treated with DOCA exhibited a decrease in HPCs in peripheral blood but not bone marrow associated with decreased telomerase activity. Conclusions— This is the first study to demonstrate that mineralocorticoid excess, either endogenous or exogenous, results in reduction in HPCs. These data suggest that mineralocorticoids may induce accelerated senescence of progenitor cells, leading to their reduced survival and decline in numbers.

Background— When advanced, heart failure with preserved ejection fraction (HFpEF) is readily apparent. However, diagnosis of earlier disease may be challenging because exertional dyspnea is not specific for heart failure, and biomarkers and hemodynamic indicators of volume overload may be absent at rest. Methods and Results— Patients with exertional dyspnea and ejection fraction >50% were referred for hemodynamic catheterization. Those with no significant coronary disease, normal brain natriuretic peptide assay, and normal resting hemodynamics (mean pulmonary artery pressure <25 mm Hg and pulmonary capillary wedge pressure [PCWP] <15 mm Hg) (n=55) underwent exercise study. The exercise PCWP was used to classify patients as having HFpEF (PCWP ≥25 mm Hg) (n=32) or noncardiac dyspnea (PCWP <25 mm Hg) (n=23). At rest, patients with HFpEF had higher resting pulmonary artery pressure and PCWP, although all values fell within normal limits. Exercise-induced elevation in PCWP in HFpEF was confirmed by greater increases in left ventricular end-diastolic pressure and was associated with blunted increases in heart rate, systemic vasodilation, and cardiac output. Exercise-induced pulmonary hypertension was present in 88% of patients with HFpEF and was related principally to elevated PCWP, as pulmonary vascular resistances dropped similarly in both groups. Exercise PCWP and pulmonary artery systolic pressure were highly correlated. An exercise pulmonary artery systolic pressure ≥45 mm Hg identified HFpEF with 96% sensitivity and 95% specificity. Conclusions— Euvolemic patients with exertional dyspnea, normal brain natriuretic peptide, and normal cardiac filling pressures at rest may have markedly abnormal hemodynamic responses during exercise, suggesting that chronic symptoms are related to heart failure. Earlier and more accurate diagnosis using exercise hemodynamics may allow better targeting of interventions to treat and prevent HFpEF progression.

Background— Mortality among patients with heart failure is high. Though individual biomarkers have been investigated to determine their value in mortality risk prediction, the role of a multimarker strategy requires further evaluation. Methods and Results— Olmsted County residents presenting with heart failure from July 2004 to September 2007 were recruited to undergo biomarker measurement. We investigated whether addition of C-reactive protein, B-type natriuretic peptide, and troponin T to a model including established risk indicators improved 1-year mortality risk prediction using the c statistic, integrated discrimination improvement, and net reclassification improvement. Among 593 participants, the mean age was 76.4 years, and 48% were men. After 1 year of follow-up, 122 (20.6%) participants had died. Patients with C-reactive protein (<11.8 mg/L), B-type natriuretic peptide (<350 pg/mL), and troponin T (≤0.01 ng/mL) less than the median had low 1-year mortality (3.3%), whereas those with 2 or 3 biomarkers greater than the median had markedly increased mortality (30.8% and 35.5%, respectively). The addition of 2 or more biomarkers to the model offered greater improvement in 1-year mortality risk prediction than use of a single biomarker. The combination of C-reactive protein and B-type natriuretic peptide resulted in an increase in the c statistic from 0.757 to 0.810 ( P <0.001), an integrated discrimination improvement gain of 7.1% ( P <0.001), and a net reclassification improvement of 22.1% ( P <0.001). Use of all 3 biomarkers offered no incremental gain (integrated discrimination improvement gain 0.7% versus C-reactive protein+B-type natriuretic peptide, P =0.065). Conclusions— Biomarkers improved 1-year mortality risk prediction beyond established indicators. The use of a 2-biomarker combination was superior to a single biomarker in risk prediction, though addition of a third biomarker conferred no added benefit.

Alternative RNA splicing may provide unique opportunities to identify drug targets and therapeutics. We identified an alternative spliced transcript for B-type natriuretic peptide (BNP) resulting from intronic retention. This transcript is present in failing human hearts and is reduced following mechanical unloading. The intron-retained transcript would generate a unique 34 amino acid (aa) carboxyl terminus while maintaining the remaining structure of native BNP. We generated antisera to this carboxyl terminus and identified immunoreactivity in failing human heart tissue. The alternatively spliced peptide (ASBNP) was synthesized and unlike BNP, failed to stimulate cGMP in vascular cells or vasorelax preconstricted arterial rings. This suggests that ASBNP may lack the dose-limiting effects of recombinant BNP. Given structural considerations, a carboxyl-terminal truncated form of ASBNP was generated (ASBNP.1) and was determined to retain the ability of BNP to stimulate cGMP in canine glomerular isolates and cultured human mesangial cells but lacked similar effects in vascular cells. In a canine-pacing model of heart failure, systemic infusion of ASBNP.1 did not alter mean arterial pressure but increased the glomerular filtration rate (GFR), suppressed plasma renin and angiotensin, while inducing natriuresis and diuresis. Consistent with its distinct in vivo effects, the activity of ASBNP.1 may not be explained through binding and activation of NPR-A or NPR-B. Thus, the biodesigner peptide ASBNP.1 enhances GFR associated with heart failure while lacking the vasoactive properties of BNP. These findings demonstrate that peptides with unique properties may be designed based on products of alternatively splicing.

Background— In contrast to the wealth of data on isolated systolic hypertension involving the systemic circulation in the elderly, much less is known about age-related change in pulmonary artery systolic pressure (PASP) and its prognostic impact in the general population. We sought to define the relationship between PASP and age, to evaluate which factors influence PASP, and to determine whether PASP is independently predictive of mortality in the community. Methods and Results— A random sample of the Olmsted County, Minn, general population (n=2042) underwent echocardiography and spirometry and was followed up for a median of 9 years. PASP was measured from the tricuspid regurgitation velocity. Left ventricular diastolic pressure was estimated with Doppler echocardiography (E/e′ ratio), and arterial stiffening was assessed from the brachial artery pulse pressure. Among 1413 subjects (69%) with measurable PASP (age, 63±11 years; 43% male), median PASP was 26 mm Hg (25th to 75th percentile, 24 to 30 mm Hg) and increased with age ( r =0.31, P <0.001). Independent predictors of PASP were age, pulse pressure, and mitral E/e′ (all P ≤0.003). Increasing PASP was associated with higher mortality (hazard ratio, 2.73 per 10 mm Hg; P <0.001). In subjects without cardiopulmonary disease (any heart failure, coronary artery disease, hypertension, diabetes mellitus, or chronic obstructive lung disease), the age-adjusted hazard ratio was 2.74 per 10 mm Hg ( P =0.016). Conclusions— We provide the first population-based evidence of age-related increase in pulmonary artery pressure, its association with increasing left heart diastolic pressures and systemic vascular stiffening, and its negative impact on survival. Pulmonary artery pressure may serve as a novel cardiovascular risk factor and potential therapeutic target.

Background— Advanced glycation end products (AGEs) are believed to increase left ventricular (LV) and vascular stiffness, in part via cross-linking proteins. We determined whether and where AGEs were increased in elderly hypertensive nondiabetic dogs and whether an AGE cross-link breaker (ALT-711) improved vascular or ventricular function. Methods and Results— Elderly dogs with experimental hypertension (old hypertensives [OH]) were randomized to receive ALT-711 (OH+ALT group; n=11; 1 mg/kg PO) or not (OH group; n=11) for 8 weeks. Conscious blood pressure measurements (weekly), echocardiography (week 8), and anesthetized study (week 8) with LV pressure–volume analysis and aortic pressure–dimension and pressure–flow assessment over a range of preloads and afterloads were performed. In LV and aorta from OH, OH+ALT, and young normal dogs, AGE content (immunohistochemistry and Western analysis for N ε -(carboxymethyl)lysine [CML]) was assessed. Aortic CML content was markedly increased in OH and OH+ALT dogs compared with young normal dogs. CML was localized to aortic and aortic vasa vasorum smooth muscle but not to collagen or elastin. CML was essentially undetectable in young normal, OH, or OH+ALT myocardium but was visible in large vessels in the LV. ALT-711 therapy was associated with lower blood pressure and pulse pressure, decreased systemic vascular resistance, increased aortic distensibility and arterial compliance, and, notably, significant aortic dilatation. Neither LV systolic nor diastolic function was different in OH+ALT versus OH dogs. Conclusions— In elderly hypertensive canines, AGE accumulation and AGE cross-link breaker effects were confined to the vasculature without evidence of myocardial accumulation or effects. The lack of AGE accumulation in collagen-rich areas suggests that the striking vascular effects may be mediated by mechanisms other than collagen cross-linking.

Background— Tumor necrosis factor-α (TNFα), an inflammatory cytokine, was reported to be elevated in trials of heart failure (HF) with reduced ejection fraction (EF) and associated with mortality. Whether this is true for HF with preserved EF is unknown, and community data are lacking. We evaluated the distribution of TNFα, its association with baseline characteristics and mortality, and its benefit in assessing risk in community HF patients. Methods and Results— Olmsted County residents with active HF from July 2004 to March 2007 (n=486; mean age, 76.7 years; EF ≥50%, 55%) were prospectively recruited. Clinical characteristics and TNFα were measured. Elevated TNFα (more than the assay limit of normal of 2.8 pg/mL) was present in 143 (29%). Higher TNFα was associated with decreased creatinine clearance, nonsmoking status, anemia, and greater comorbidity ( P trend <0.05 for all). Mortality increased with increasing TNFα ( P =0.016), with 1-year mortality estimates of 16%, 18%, 23%, and 32% from the lowest to highest quartile, respectively. After adjustment for age, sex, and EF, the hazard ratios for death were 1.24, 1.37, and 1.90 from the second to the highest TNFα quartile, respectively ( P trend =0.007). TNFα contributed to risk assessment as indicated by increases in the area under the receiver operating characteristic curves in all models examined ( P <0.05 for all). Results did not differ by EF ( P =0.60 interaction term of TNFα and EF). Conclusions— TNFα was elevated in a large portion of community HF patients, was associated with a large decrease in survival, and provided a significant incremental increase in risk assessment above established indicators. TNFα is useful for risk assessment in HF patients with preserved and reduced EF.

Background— Altered titin isoforms may modify cardiac function in heart failure (HF), but the nature of isoform switches and associated functional implications are not well defined. Limited studies have reported an increased compliant isoform (N2BA) expression in human systolic HF. Titin may also modulate stretch-regulated responses such as myocardial natriuretic peptide production. Methods and Results— We characterized titin isoform expression and extracellular matrix in all 4 cardiac chambers and the left ventricular (LV) epicardium and endocardium in normal dogs (n=6) and those with HF (n=6) due to tachypacing and characterized functional implications at the LV myofiber and chamber level. Recognizing the potential for uncoupling of the extracellular matrix and cardiomyocyte in tachypacing, myocardial natriuretic peptide production, a molecular marker of stretch-regulated responses, was also assessed. All chambers were dilated in HF, but the extracellular matrix was not increased. HF dogs had markedly lower N2BA in the atria and right ventricle. In failing LVs, N2BA was decreased only in the epicardium, where myofiber passive stiffness was increased. However, LV chamber mechanics were driven by the marked LV dilatation, with no increase in LV diastolic stiffness. Natriuretic peptide concentrations increased markedly in the endocardium in relation to increases in LV wall stress. Conclusions— Tachypacing HF is characterized by decreases in compliant titin isoform expression in the atria, right ventricle, and LV epicardium. However, LV chamber mechanics are principally determined by geometric and extracellular matrix changes rather than titin-based myofiber stiffness in this model. Stretch-regulated myocardial responses (natriuretic peptide production) appeared intact, suggesting that the mechanotransduction role of titin was not impaired in HF.

Background— Mortality in patients with heart failure (HF) remains high, but causes of death are incompletely defined. As HF is a heterogeneous syndrome categorized according to the ejection fraction (EF), the association between EF and causes of death is important, yet elusive. Methods and Results— Community subjects with HF were classified according to the preserved (≥50%) and the reduced EF (<50%). Deaths were classified as due to coronary heart disease and other cardiovascular and noncardiovascular diseases. Among 1063 persons with HF, 45% had preserved EF with fewer cardiovascular risk factors and less coronary disease than those with reduced EF. At 5 years, survival was 45% (95% CI, 43% to 49%), and 43% of the deaths were noncardiovascular. The leading cause of death in subjects with preserved EF was noncardiovascular disease (49%) versus coronary heart disease (43%) for subjects with reduced EF. The proportion of cardiovascular deaths decreased from 69% in 1979–1984 to 40% in 1997–2002 ( P =0.007) among subjects with preserved EF, which is in contrast to a modest change among those with reduced EF (77% to 64%, P =0.08). Advanced age, male sex, diabetes, smoking, and kidney disease were associated with an increased risk of all-cause and cardiovascular deaths. After adjustment, preserved EF was associated with a lower risk of cardiovascular death but not all-cause death. Conclusions— Community subjects with HF experience a persistently high mortality, and a large proportion of deaths is noncardiovascular. Cardiovascular disease before death is less in subjects with preserved EF, and they are less likely to experience cardiovascular deaths compared with those with reduced EF. In those with preserved EF, the proportion of cardiovascular deaths declined over time.