Burnett, John C.

BACKGROUND Although recent investigations report the elevation of plasma endothelin (ET) in congestive heart failure (CHF), it remains unclear if this elevation is that of the biologically active peptide ET-1 or of its precursor big-ET. Furthermore, it is unclear if such elevation is associated with increased myocardial ET and if the molecular form from cardiac tissue is altered ET. Last, it remains to be established whether circulating ET is increased at the earliest stage of CHF in patients with asymptomatic left ventricular dysfunction and correlates with the magnitude of ventricular dysfunction. METHODS AND RESULTS The present study was designed to investigate concentrations and molecular forms of ET in plasma and cardiac tissue in healthy subjects and CHF patients with New York Heart Association (NYHA) class I through IV using cardiac radionuclide angiogram, cardiac myocardial biopsy, radioimmunoassay, gel permeation chromatography (GPC), and immunohistochemical staining (IHCS). Plasma ET was increased only in patients with moderate (NYHA class III) or severe (NYHA class IV) CHF compared with healthy subjects and individuals with asymptomatic (NYHA class I) or mild (NYHA class II) CHF. The elevation of circulating ET in CHF showed a negative correlation with left ventricular ejection fraction and cardiac index and a positive correlation with functional class and left ventricular end-diastolic volume index. GPC demonstrated that immunoreactive plasma ET was ET-1 in healthy subjects and both mature ET-1 and its precursor big-ET in severe CHF patients, with big-ET the predominant molecular form. Cardiac tissue concentrations and IHCS revealed ET presence in healthy atrial and ventricular tissue, which were not different in severe CHF. GPC revealed that the molecular form of cardiac ET was ET-1 in both healthy and CHF hearts. CONCLUSIONS The present study establishes for the first time that the elevation of plasma ET in severe human CHF represents principally elevation of big-ET. Second, ET is present in healthy and failing myocardia, and its activity by both immunohistochemistry and radioimmunoassay is not changed in CHF. Furthermore, the elevated plasma ET is characteristic of severe CHF and not asymptomatic or mild CHF. In addition, the degree of plasma elevation of ET correlates with the magnitude of alterations in cardiac hemodynamics and functional class. The present study confirms and extends previous investigations of ET in human CHF and establishes the evolution of circulating and local cardiac ET in the spectrum of human CHF.

Background — The role of load versus angiotensin II (Ang II) and endothelin-1 (ET) in the pathogenesis of hypertensive heart disease is controversial. We sought to determine whether alterations in cardiac structure and function due to hypertension (HTN) were dependent on Ang II or ET activation. Methods and Results — Bilateral renal wrapping to produce HTN (n=12) or sham surgery (n=6) was performed in adult dogs. Weekly blood pressure, plasma renin activity, Ang II, ET, and catecholamines were measured. Systolic (end-systolic elastance, Ees) and diastolic (τ) function were assessed in sham and HTN dogs at 5 (HTN-5wk) or 12 (HTN-12wk) weeks. Ang II and ET were assayed in the left ventricle (LV) and kidney. Mean arterial pressure was higher in renal wrap dogs at week 1 (* P <0.05 versus controls: 139±4* versus 123±4 mm Hg), week 5 (174±7* versus 124±4 mm Hg), and week 12 (181±12* versus 124±4 mm Hg). LV mass index was increased in HTN-5wk (22%*) and HTN-12wk (39%*). LV fibrosis was increased in HTN-12wk. Ees was preserved in HTN-5wk and HTN-12wk. τ was increased in HTN-5wk (50±3* ms) and HTN-12wk (62±10* ms) dogs compared with sham (41±2 ms). Plasma Ang II, ET, catecholamines, and plasma renin activity were unchanged during the progressive HTN. Ang II and ET in LV and kidney were not different from controls. Conclusions — Systemic HTN induces LV hypertrophy, myocardial fibrosis, and isolated diastolic dysfunction in the absence of local or systemic activation of Ang II or ET. These findings suggest that load is the prevailing stimulus for the structural and functional changes associated with early hypertensive heart disease.

BACKGROUND The endothelium possesses the ability to modulate vascular tone by the release of vasodilators and vasoconstrictors, among them endothelium-derived relaxing factor (EDRF) and endothelin (ET). Abnormalities in EDRF generation have been demonstrated in various cardiovascular pathophysiological states. Moreover, a twofold increase in plasma ET concentration was reported in these disease states. Recent in vitro studies have suggested the interaction between these two endothelium-derived substances, suggesting that imbalance between the two may contribute to alternation in vascular tone characteristic of these disease states. Thus, the hypothesis of this study was that inhibition of endogenous EDRF will enhance the vasoconstrictor response to a twofold increase in plasma ET concentrations. METHODS AND RESULTS Experiments were conducted in three groups of anesthetized dogs. In group 1, ET-1 was infused intravenously to double circulating ET concentrations. Group 2 received both ET and NG-monomethyl L-arginine (L-NMMA), a competitive inhibitor of EDRF generation, and group 3 received a continuous infusion of L-NMMA alone. Twofold increase in plasma ET concentrations was characterized by an increase in systemic and renal vasoconstriction. The inhibition of EDRF markedly enhanced the vasoconstriction to ET specifically involving the systemic, pulmonary, coronary, and renal arterial circulations. CONCLUSIONS The present study demonstrates that inhibition of endogenous EDRF augments the vasoconstrictor property of ET and supports a functional role for the balance between endothelium-derived vasodilating and vasoconstricting factors in the regulation of vascular tone.

Background— Alterations at the level of the coronary circulation with aging may play an important role in the evolution of age-associated changes in left ventricular (LV) fibrosis and function. However these age-associated changes in the coronary vasculature remain poorly defined primarily due to the lack of high resolution imaging technologies. The current study was designed to utilize cardiac micro–computed tomography (micro-CT) technology as a novel imaging strategy, to define the 3-dimensional coronary circulation in the young and aged heart and its relationship to LV fibrosis and function. Methods and Results— Young (2 months old; n=10) and aged (20 months old; n=10) Fischer rats underwent cardiac micro-CT imaging as well as echocardiography, blood pressure, and fibrosis analysis. Importantly, when indexed to LV mass, which increased with age, the total and intramyocardial vessel volumes were lower, whereas the epicardial vessel volume, with and without indexing to LV mass, was significantly higher in the aged hearts compared with the young hearts. Moreover, the aged hearts had a significantly lower percentage of intramyocardial vessel volume and a significantly higher percentage of epicardial vessel volume, when normalized to the total vessel volume, compared with the young hearts. Further, the aged hearts had significant LV fibrosis and mild LV dysfunction compared with the young hearts. Conclusions— This micro-CT imaging study reports the reduction in normalized intramyocardial vessel volume within the aged heart, in association with increased epicardial vessel volume, in the setting of increased LV fibrosis, and mild LV dysfunction.

Background— In vitro studies suggest that phosphorylation of titin reduces myocyte/myofiber stiffness. Titin can be phosphorylated by cGMP-activated protein kinase. Intracellular cGMP production is stimulated by B-type natriuretic peptide (BNP) and degraded by phosphodiesterases, including phosphodiesterase-5A. We hypothesized that a phosphodiesterase-5A inhibitor (sildenafil) alone or in combination with BNP would increase left ventricular diastolic distensibility by phosphorylating titin. Methods and Results— Eight elderly dogs with experimental hypertension and 4 young normal dogs underwent measurement of the end-diastolic pressure-volume relationship during caval occlusion at baseline, after sildenafil, and BNP infusion. To assess diastolic distensibility independently of load/extrinsic forces, the end-diastolic volume at a common end-diastolic pressure on the sequential end-diastolic pressure-volume relationships was measured (left ventricular capacitance). In a separate group of dogs (n=7 old hypertensive and 7 young normal), serial full-thickness left ventricular biopsies were harvested from the beating heart during identical infusions to measure myofilament protein phosphorylation. Plasma cGMP increased with sildenafil and further with BNP (7.31±2.37 to 26.9±10.3 to 70.3±8.1 pmol/mL; P <0.001). Left ventricular diastolic capacitance increased with sildenafil and further with BNP (51.4±16.9 to 53.7±16.8 to 60.0±19.4 mL; P <0.001). Changes were similar in old hypertensive and young normal dogs. There were no effects on phosphorylation of troponin I, troponin T, phospholamban, or myosin light chain-1 or -2. Titin phosphorylation increased with sildenafil and BNP, whereas titin-based cardiomyocyte stiffness decreased. Conclusion— Short-term cGMP-enhancing treatment with sildenafil and BNP improves left ventricular diastolic distensibility in vivo, in part by phosphorylating titin.

Impaired renal function with loss of nephron number in chronic renal disease (CKD) is associated with increased cardiovascular morbidity and mortality. However, the structural and functional cardiac response to early and mild reduction in renal mass is poorly defined. We hypothesized that mild renal impairment produced by unilateral nephrectomy (UNX) would result in early cardiac fibrosis and impaired diastolic function, which would progress to a more global left ventricular (LV) dysfunction. Cardiorenal function and structure were assessed in rats at 4 and 16 wk following UNX or sham operation (Sham); ( n = 10 per group). At 4 wk, blood pressure (BP), aldosterone, glomerular filtration rate (GFR), proteinuria, and plasma B-type natriuretic peptide (BNP) were not altered by UNX, representing a model of mild early CKD. However, UNX was associated with significantly greater LV myocardial fibrosis compared with Sham. Importantly, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining revealed increased apoptosis in the LV myocardium. Further, diastolic dysfunction, assessed by strain echocardiography, but with preserved LVEF, was observed. Changes in genes related to the TGF-β and apoptosis pathways in the LV myocardium were also observed. At 16 wk post-UNX, we observed persistent LV fibrosis and impairment in LV diastolic function. In addition, LV mass significantly increased, as did LVEDd, while there was a reduction in LVEF. Aldosterone, BNP, and proteinuria were increased, while GFR was decreased. The myocardial, structural, and functional alterations were associated with persistent changes in the TGF-β pathway and even more widespread changes in the LV apoptotic pathway. These studies demonstrate that mild renal insufficiency in the rat results in early cardiac fibrosis and impaired diastolic function, which progresses to more global LV remodeling and dysfunction. Thus, these studies importantly advance the concept of a kidney-heart connection in the control of myocardial structure and function.

Renal aging is characterized by structural changes in the kidney including fibrosis, which contributes to the increased risk of kidney and cardiac failure in the elderly. Studies involving healthy kidney donors demonstrated subclinical age-related nephropathy on renal biopsy that was not detected by standard diagnostic tests. Thus there is a high-priority need for novel noninvasive biomarkers to detect the presence of preclinical age-associated renal structural and functional changes. C-type natriuretic peptide (CNP) possesses renoprotective properties and is present in the kidney; however, its modulation during aging remains undefined. We assessed circulating and urinary CNP in a Fischer rat model of experimental aging and also determined renal structural and functional adaptations to the aging process. Histological and electron microscopic analysis demonstrated significant renal fibrosis, glomerular basement membrane thickening, and mesangial matrix expansion with aging. While plasma CNP levels progressively declined with aging, urinary CNP excretion increased, along with the ratio of urinary to plasma CNP, which preceded significant elevations in proteinuria and blood pressure. Also, CNP immunoreactivity was increased in the distal and proximal tubules in both the aging rat and aging human kidneys. Our findings provide evidence that urinary CNP and its ratio to plasma CNP may represent a novel biomarker for early age-mediated renal structural alterations, particularly fibrosis. Thus urinary CNP could potentially aid in identifying subjects with preclinical structural changes before the onset of symptoms and disease, allowing for the initiation of strategies designed to prevent the progression of chronic kidney disease particularly in the aging population.

Background— The angiotensin II type 1 receptor (AT1R) plays a key role in regulating cardiorenal function. Classic “unbiased” AT1R antagonists block receptor coupling to both G αq and ß-arrestin–mediated signals, which desensitize G-protein signaling as well as transduce G-protein–independent signals. TRV120027 is a novel ß-arrestin–biased AT1R ligand, which engages ß-arrestins while blocking G-protein signaling. At the AT1R, TRV120027 can inhibit angiotensin II–mediated vasoconstriction, whereas, through ß-arrestin coupling, increase cardiomyocyte contractility. We defined for the first time the acute cardiorenal actions of TRV120027 in healthy and heart failure (HF) canines. Methods and Results— Healthy and HF canines (induced by tachypacing) were anesthetized. After instrumentation and equilibration, a 30-minute baseline clearance was performed, followed by further clearance with escalating doses of intravenous TRV120027 (0.01, 0.1, 1, 10, and 100 μg/kg per minute) and a 30-minute washout. In healthy canines, TRV120027 decreased pulmonary capillary wedge pressure and systemic and renal vascular resistances, while increasing cardiac output, renal blood flow, glomerular filtration rate, and urinary sodium excretion. In HF canines, TRV120027 decreased mean arterial pressure, right atrial pressure, and pulmonary capillary wedge pressure, systemic and renal vascular resistances and increased cardiac output and renal blood flow. Glomerular filtration rate and urinary sodium excretion were maintained. Conclusions— We report for the first time the cardiorenal actions of the novel ß-arrestin–biased AT1R ligand TRV120027. In both normal and HF canines, TRV120027 demonstrated cardiac unloading actions while preserving renal function. With this beneficial pharmacological profile, TRV120027 represents a novel strategy for the treatment of HF.

Despite recent pharmacological advances in heart failure therapy, mortality from acute decompensated heart failure remains high. Conventional therapies are often insufficient to address the complex interplay between structural, functional, neurohumoral, and renal mechanisms involved in the heart failure syndrome. The natriuretic peptide system, however, offers a unique pleiotropic strategy which could bridge this gap in heart failure therapy. Exogenous administration of native A-type and B-type natriuretic peptides has been met with both success and limitations, and despite the limitations, remains a worthwhile endeavor. Alternatively, synthetic modification to create “designer” chimeric peptides holds the possibility to extend both the application and therapeutic benefits possible with a natriuretic peptide based approach. Herein we describe the development of natriuretic peptide based heart failure therapies, including the design, rationale, and preliminary studies of the novel chimeric peptides CD-NP and CU-NP.

Recent studies demonstrate that endothelin, a potent endogenous vasoconstrictor peptide, circulates in plasma of normal animals and humans. However, the role of this peptide in pathophysiological states remains unclear. The present study was designed to test the hypothesis that circulating endothelin concentrations are increased in experimental congestive heart failure (CHF), a pathophysiological state characterized by activation of vasoconstrictor mechanisms. In anesthetized dogs with CHF produced by 8 days of rapid ventricular pacing (n = 28), circulating plasma endothelin was increased compared with values for normal controls (n = 28; 20.4 +/- 1.4 versus 9.7 +/- 0.9 pg/ml, respectively; p less than 0.0001). A plasma endothelin level of more than 14.0 was a sensitive and specific indicator of significant CHF. Moreover, within the group with experimental CHF, right atrial pressure and pulmonary capillary wedge pressure correlated independently with circulating endothelin levels. Based on recent studies demonstrating the physiological actions of twofold increases in circulating endothelin, as observed in the present study, a possible role for endothelin in the pathophysiology of CHF is advanced.