Sharada L. Truter

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.

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.

Abnormal gene expression of cardiac fibroblasts in experimental aortic regurgitation.

&NA; Extensive primary fibrosis precedes heart failure and death in experimental chronic aortic regurgitation. To seek the molecular basis for this observation, this study analyzed the RNA pool for genes that are up- or downregulated in aortic regurgitation fibroblasts. Differential display reverse transcriptase polymerase chain reaction was used to compare RNA extracted from cardiac fibroblasts isolated from three healthy New Zealand white rabbits and from three with aortic regurgitation. Using two base anchoring oligo d(T) primers (T11VN) together with arbitrary upstream primers, numerous differences in normal versus aortic regurgitation gene expression were apparent on differential display reverse transcriptase polymerase chain reaction. The aortic regurgitation cell cultures showed numerous differentially up- and downregulated genes compared with cell cultures of normal cardiac fibroblasts. The results showed that pathologic fibrosis in chronic experimental aortic regurgitation is associated with abnormal cardiac fibroblast gene expression, which may be pathogenic for the fibrous lesion.

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.

Vesnarinone-mediated alterations of gene expression in cardiac fibroblasts from aortic regurgitant hearts.

Pathologic fibrosis precedes heart failure (CHF) and death in experimental aortic regurgitation (AR). Vesnarinone, a positively inotropic quinolone derivative, suppresses survival of fibroblasts (CF) from hearts with chronic experimental AR. To explore further the potential effects of vesnarinone on cardiac fibrosis in AR, we tested the hypothesis that vesnarinone suppresses gene expression induced by AR in CF. Differentially expressed genes were isolated by suppression subtractive hybridization (SSH) in CF from hearts of 2 New Zealand White rabbits with surgically induced AR compared with 2 normal rabbits. In cultured AR-CF treated with and without vesnarinone (4 doses, including the dose that had caused maximal survival suppression in cultured AR-CF), drug effect was assessed on expression of genes found to be up-regulated by AR. SSH, reverse Northern analysis, and Northern analysis indicated that at doses several orders of magnitude lower than those used for treatment in CHF vesnarinone significantly down-regulated 2 genes (thrombospondin 1, annexin II) up-regulated by AR. The study confirmed earlier findings of AR-mediated alteration in expression of genes that code for noncollagen extracellular matrix (ECM) proteins. Thus, in CF conditioned by exposure to AR, vesnarinone at relatively low doses suppresses genes coding for 2 noncollagen ECM proteins up-regulated by AR. These pharmacologic effects may underlie potentially therapeutic mitigation of fibrosis by vesnarinone.

Pathophysiology of Heart Failure in Regurgitant Valvular Diseases: Relation to Ventricular Dysfunction and Clinical Debility

Regurgitant valvular heart diseases and, most particularly, insufficiency of the aortic and/or mitral valves (AR, MR), are among the more common, and most predictable, causes of congestive heart failure. Current data suggest that these conditions also confer a proclivity for sudden death, even among asymptomatic or minimally symptomatic patients (1,2,3).

Fibronectin Gene Expression in Aortic Regurgitation: Relative Roles of Mitogen-Activated Protein Kinases

Objectives: In aortic regurgitation (AR), fibronectin (FN) expression is upregulated. This study sought to determine signal transduction pathways involved in upregulation of FN expression in AR. Methods: Cardiac fibroblasts (CF) from rabbits with surgically induced AR and matched controls (NL) were cultured and assayed for FN expression and kinase activity with and without inhibitors of kinases JNK, p38 mitogen-activated protein kinase (MAPK) and extracellular response kinase (ERK). NL CF also were subjected to cyclic strain mimicking AR for 24 h in culture with and without inhibitors. Results: AR CF exhibited 2.9-fold greater c-Jun phosphorylation (p < 0.01) and 1.5- to 2-fold greater ATF2 phosphorylation (p < 0.05–0.01) than NL. JNK and p38MAPK inhibition reduced c-Jun and ATF2 phosphorylation to NL; ERK inhibition had no effect. FN mRNA expression was similar in pattern to kinase activities. Cyclic strain in NL CF increased c-Jun phosphorylation 2-fold versus unstrained controls (p < 0.005). This was suppressed by inhibition of JNK but not p38MAPK. Conclusion: FN expression in response to the acute mechanical strain resembling AR is upregulated primarily via JNK. However, in chronic AR both JNK and p38MAPK are involved. These signaling pathways represent potential therapeutic targets for normalizing extracellular matrix (ECM) composition and contractile force transmission, believed to be related to ECM composition/organization, in AR.