Edmund M. Herrold

Myocardial Fibrosis in Chronic Aortic Regurgitation Molecular and Cellular Responses to Volume Overload

Background—Myocardial fibrosis is common in patients with chronic aortic regurgitation (AR). Experimentally, fibrosis with disproportionate noncollagen extracellular matrix (ECM) elements precedes and contributes to heart failure in AR. Method and Results—We assessed [3H]-glucosamine and [3H]-proline incorporation in ECM, variations in cardiac fibroblast (CF) gene expression, and synthesis of specific ECM proteins in CF cultured from rabbits with surgically induced chronic AR versus controls. To determine whether these variations are primary responses to AR, normal CF were exposed to mechanical strain that mimicked that of AR. Compared with normal CF, AR CF incorporated more glucosamine (1.8:1, P =0.001) into ECM, showed fibronectin gene upregulation (2.0:1, P =0.02), and synthesized more fibronectin (2:1 by Western blot, P <0.06; 1.5:1 by affinity chromatography, P =0.02). Proline incorporation was unchanged by AR (1.1:1, NS); collagen synthesis was unaffected (type I, 0.9:1; type III, 1.0:1, NS). Normal CF exposed to cyclical mechanical strain during culture showed parallel results: glucosamine incorporation increased with strain (2.1:1, P <0.001), proline incorporation was unaffected (1.1:1, NS), fibronectin gene expression (1.6:1, P =0.07) and fibronectin synthesis (Western analysis, 1.3:1, P <0.01; chromatography, 1.9:1, NS) were upregulated. Conclusions—In AR, CF produce abnormal proportions of noncollagen ECM, specifically fibronectin, with relatively little change in collagen synthesis. At least in part, this is a primary response to strain imposed on CF by AR. Further study must relate these findings to the pathogenesis of heart failure in AR.

External Imaging of Atherosclerosis in Rabbits Using an 123I‐Labeled Synthetic Peptide Fragment

The oligopeptide fragment of apolipoprotein B, SP‐4, has demonstrated pronounced uptake in the healing edges of balloon‐injured rabbit aortic endothelium. To assess 123I‐labeled SP‐4 for identification of atherosclerotic plaques by gamma camera imaging, 14 Watanabe heritable hyperlipidemic (WHHL) and 5 normal rabbits were imaged 5 minutes and 12 and 24 hours after intravenous injection of123I‐SP‐4. In addition, two WHHL and two normal rabbits were injected with 125I‐SP‐4 for autoradiography. Twelve of the 14 WHHL, but none of the normal, rabbits had visually apparent focal radioiodine accumulation in the region of the aorta. Focus‐to‐lung and focus‐to‐heart count ratios were 2.4 ± 1.3 and 1.0 ± 0.4, respectively. Five of the visually positive WHHL rabbits were reimaged 4 and 8 weeks later with 123I‐NaI and 123I‐SP‐2 (an apo E peptide), respectively, as negative controls. Perceptible, but faint, aortic localization of 123I‐NaI and of 123I‐SP‐2 was seen in only one animal each. The distributions of atherosclerotic lesions on photographs of the opened WHHL aortas and of film blackening on 125I‐SP‐4 autoradiograms were identical. In contrast, the two normal rabbit aortas did not exhibit plaques on photographs or film blackening on autoradiograms. Thus, in an animal model closely simulating human atherosclerotic disease, SP‐4 localizes specifically in aortic atherosclerotic lesions.

Differential Expression of Matrix Metalloproteinases and Tissue Inhibitors and Extracellular Matrix Remodeling in Aortic Regurgitant Hearts

Objectives: Myocardial fibrosis in experimental aortic regurgitation (AR) features abnormal fibronectin with normal collagen content, but the relevant degradative processes have not been assessed. Methods: To elucidate these degradative processes, mRNA (Northern) and protein levels (Western) of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), as well as MMP activity (zymography), were measured in cardiac fibroblasts (CF) from New Zealand white rabbits with experimental AR paired with normals (NL). Collagen and fibronectin were quantified by immunohistochemical staining. Results: In AR CF versus NL CF, MMP-2 and -14 mRNA and protein were increased (both p < 0.005), while TIMPs 1–3 were slightly decreased (p < 0.05–0.005; TIMP-4 undetectable). Gelatinase activity in AR CF was 1.7 times that in NL CF (p < 0.005); fibronectinase activity was unaffected. The Jun N-terminal kinase (JNK) inhibitor SP600125 suppressed MMP-2 protein (0.4-fold, p < 0.05) and mRNA (0.7-fold, p < 0.005) in AR CF; MMP-2 levels in NL CF were unaffected. AR MMP-9 mRNA, protein and activity were low and indistinguishable from NL. In left ventricular tissue, fibronectin was increased 1.9-fold (AR vs. NL, p < 0.05). Total AR collagen was indistinguishable from NL, but the collagen III to collagen I isoform ratio decreased (0.4-fold, p < 0.05). Conclusions: Collagen is relatively deficient in AR fibrosis, due at least in part to upregulated MMPs and downregulated TIMPs; fibronectinase is unaltered. JNK-dependent regulation may stimulate both MMP-2 and fibronectin expression in AR, providing a potential therapeutic target.

Computer-assisted analysis of Holter recordings.

Early work in radiotelemetry and signal processing by Holter’ made it possible to detect changes in heart rate and cardiac rhythm in ambulatory subjects. Heart rhythm was identified by cycle-length-dependent superimposition of electrocardiographic complexes, with a 60-fold ratio of playback to recording speed to allow data analysis in compressed time. By 1964, Gilson et al. were able to report that simple arrhythmias, brief changes in electrocardiographic morphology, and a wide range of artifacts could be detected by these methods in apparently normal subjects.2 Useful association of transient arrhythmias with symptoms was observed by Corday et al. in patients studied with 10-hour continuous recordings,’ and limitations and advantages of early ambulatory recording methods were reviewed by Hinkle e f al. in 1967.4 Advancing technology soon made it practical to acquire longer samples of analog electrocardiographic data on a lightweight, portable, battery-powered recorder, using reel-to-reel tape as the storage medium. The advantage of extending continuous electrocardiographic monitoring to 24 hours to fully characterize highly sporadic and variable ambulatory rhythms was demonstrated by Lopes et al.’ While more prolonged recording periods may further increase the detection of sporadic arrhythmias,6 which may demonstrate striking spontaneous and activity-related ~ a r i a b i l i t y , ~ . ~ 24-hour continuous recordings have evolved as the most widely used compromise between technology and convenience in ambulatory populations.

Volume overload related shape change limits mass increase with wall thickening but only minimally reduces wall stress

A mathematical model was used to determine the influence of ellipsoid to spheroid left ventricular shape transformation, often seen during chronic volume overload, on ventricular mass, wall thickness, and regional wall stress. Simulated shape change predicted a mass decrease of 9% and a decrease in the maximum circumferential stress in comparison to stress normalizing hypertrophy. Both the simulation and clinical studies showed that apical wall stresses increased to 200-250% of normal with shape transformation. It is concluded that shape change offers a modest advantage of providing increased wall thickness for a given wall mass, and a reduction in maximum circumferential stress, but at the expense of greatly increased apical wall stresses, which may limit apical contractility.<<ETX>>

Cellular response of human cardiac fibroblasts to mechanically simulated aortic regurgitation.

Myocardial fibrosis has been identified in biopsy specimens from catheterization and valve replacement surgery in patients with severe chronic aortic regurgitation (AR). While characterization of these extracellular matrix (ECM) alterations has been incomplete in humans, fibrosis also has been identified in chronic severe experimentally created AR, in which ECM composition features abnormal fibronectin/glycoprotein production, with normal collagen content. Virtually identical ECM variations have been induced when normal rabbit cardiac fibroblasts (CF) are subjected in culture to cyclic mechanical strain mimicking that found in the left ventricle (LV) in severe AR. To determine whether the changes seen experimentally can be extrapolated to humans, we exposed normal human CF in culture to the mechanical strain employed in the experimental model to simulate severe AR (n = 3 replications from 1 patient). CF were isolated from epicardial biopsy distant from diseased coronary arteries in a 38-year-old man with normal LV function and without prior myocardial infarction who was undergoing elective coronary artery bypass grafting. Gelatin Sepharose affinity chromatography (GSAC) and Western analysis were used to compare fibronectin expression in strained versus nonstrained normal human CF in tissue culture; Western analysis was used to compare type I collagen production. In AR-strained CF, fibronectin synthesis nominally increased [av 38% (Western) and 45% (GSAC)] relative to control; type I collagen synthesis was virtually unchanged. These results simulate those found experimentally and suggest that human CF, like rabbit CF, manifest abnormal compositional distribution of ECM proteins in AR.

Prognostication in 3-Vessel Coronary Artery Disease Based on Left Ventricular Ejection Fraction During Exercise Influence of Coronary Artery Bypass Grafting

BACKGROUND Previous data indicate that left ventricular ejection fraction (LVEF) provides prognostic information among patients with coronary artery disease (CAD), but the value of such testing specifically for defining benefits of coronary artery bypass grafting (CABG) may relate to severity of exercise-inducible ischemia measured noninvasively before surgery. METHODS AND RESULTS To determine the independent prognostic importance of preoperative ischemia severity for predicting outcomes of CABG among patients with extensive CAD, we monitored 167 stable patients with angiographically documented 3-vessel CAD (average follow-up of 9 years in event-free patients) who previously had undergone rest and exercise radionuclide cineangiography. Their course was correlated with data obtained during initial radionuclide testing, coronary arteriography, and clinical evaluation at study entry. Fifty-two patients received medical treatment only, and 115 underwent CABG (44 early [</=1 month after initial study]). Multivariate Cox model analysis indicated that change (Delta) in LVEF from rest to exercise during radionuclide study was the strongest independent predictor of major cardiac events (P=0.003) before surgery and also predicted magnitude of CABG benefit (P=0.04). Patients with DeltaLVEF -8% or less derived significant survival-prolonging and event-reducing benefit from CABG performed </=1 month after initial testing (P<0.02 for cardiac death and P=0.008 for cardiac events], early CABG versus medical-treatment-only patients); similar benefits were absent among patients with DeltaLVEF more than -8%, and among those in whom CABG was deferred. CONCLUSIONS Assessment of ischemia severity based on LVEF response to exercise enables effective prognostication among patients with 3-vessel CAD and defines the likelihood of life-prolonging and event-reducing benefits from CABG.

Biodistribution and autoradiographic localization of I-125--labeled synthetic Peptide in aortic atherosclerosis in cholesterol-fed rabbits.

I-125-labeled SP4 is a synthetic oligopeptide derived from apolipoprotein B of low-density lipoprotein that has been shown to localize in atherosclerotic plaques in experimental animals. However, its biodistribution and mechanism of localization need to be further elucidated. Twenty-four cholesterol-fed (CF) and 20 normal (NL) New Zealand White rabbits were injected with I-125-SP4 and killed 15 to 30 min (6 NL; 6 CF) or 2 h (14 NL; 18 CF) later. We obtained aortic autoradiograms and activity concentrations (% injected dose/gin) in aortic segments and other tissues. The uptake of I-125-SP4 was higher in CF than in NL rabbits in all aortic segments (p < 0.05). I-125-SP4 was cleared rapidly in both CF and NL rabbits with 60 to 70% of the injected dose cleared from the blood by 1 h. No statistically significant differences in radiotracer biodistribution were observed between NL and CF rabbits although activity tended to be higher in the liver, gallbladder, and intestine in NL rabbits and in the kidney and spleen in CF rabbits. Silver grains were distributed mainly on foam cells of the fatty streaks in aortic microautoradiograms from two additional rabbits that had been injected with I-125-SP4. There were 23,518 ± 15,878 (SD) grains/mm2 in fatty plaques but only 14,669 ± 11,035 grains/mm2 in media muscle (p< 0.0001 [9 sections, 17 areas evaluated] in an atherosclerotic animal) in injected animals and 13,439 ± 5,565 grains/mm2 in media muscle (two sections, four areas) in the normal control animals (NS versus media of atherosclerotic animal). I-125-SP4 specifically localizes in aortic atherosclerotic plaques in CF rabbits. There is no significant difference in tissue distribution between normal and CF rabbits except in the aorta. Preliminarily, it appears that the site of tracer uptake is on foam cells and this suggests the possibility of relative specificity for fatty plaque.

Differential response to vesnarinone by cardiac fibroblasts isolated from normal and aortic regurgitant hearts.

Vesnarinone, a quinoline derivative with modest positive inotropic action, has been shown in several studies to benefit patients with clinical congestive heart failure. The cellular basis of its clinical benefit is not known, although the drug has several pharmacologic effects demonstrated both in isolated cardiac myocytes and in other noncardiac cell types. To investigate the possibility that the clinical benefit of vesnarinone is based, at least in part, on the inhibition of pathologic myocardial fibrosis, we examined its effects on cultured cardiac fibroblasts isolated from both normal and aortic regurgitant New Zealand White rabbit hearts. As in people, rabbits with moderate-to-severe aortic regurgitation often develop congestive heart failure that, at necropsy, is characterized by exuberant myocardial fibrosis. A dose-response curve was constructed with vesnarinone concentrations ranging from 10-4 to 10-9 mol/L. Cellular survival was decreased by exposure to nanomolar concentrations of drug but not at the higher doses tested. Fibroblasts isolated from normal hearts responded maximally at 10-7 mol/L vesnarinone, whereas fibroblasts from aortic regurgitant hearts responded maximally at 10-8 mol/L. These concentrations of drug are more than an order of magnitude lower than those believed to be associated with clinical benefit from earlier studies. Our results indicate that vesnarinone can suppress cardiac fibroblast proliferation and suggest that this action may be useful in therapies designed to prevent congestive heart failure in aortic regurgitation.

Relation of indirect vasodilator use to prognosis in patients with chronic severe mitral regurgitation.

Objectives: The relation of indirect vasodilator use to cardiac events (CE) is undefined for chronic severe nonischemic mitral regurgitation (MR). The aim of this study was to resolve this knowledge deficiency. Methods: Data from 52 consecutive patients in our prospective natural history study with isolated chronic severe nonischemic MR were assessed post hoc over 19 years to examine the relation of indirect vasodilator use to subsequent CE (death or indications for valve surgery). At entry, no patient had surgical indications, 14% had hypertension (HTN) and 7 chronically received vasodilators (5 angiotensin-converting enzyme inhibitor, 1 receptor blocker and 1 α-adrenergic blocker). CE differences were assessed by log-rank comparison of Kaplan-Meier curves. Results: During follow-up, CE included sudden death (1 patient), heart failure (7 patients), atrial fibrillation (6 patients), left ventricular (LV) systolic dimension >4.5 cm (12 patients), LV ejection fraction (EF) <60% (7 patients), right ventricular EF <35% (2 patients) and combination CE (7 patients). Overall, vasodilator use did not predict CE (not significant). However, patients without HTN had higher CE rates with vasodilators than without (p = 0.007), while those with HTN and vasodilators had lower CE rates than those without vasodilators (p = 0.04). Conclusion: Vasodilator use appears to confer no survival benefit in patients with chronic severe MR. The small number of patients with HTN precludes conclusions about modulation of vasodilator effect by HTN. Randomized trials are needed to conclusively evaluate this association.