Age-associated changes in cardiac matrix and integrins

Abstract

The progressive shift from young age to senescence is characterized by structural and functional changes in the cardiac extracellular matrix (ECM), which supports and aligns myocytes and blood vessels, and maintains myocardial mass, structure and function. As cardiac function declines with advancing age, ECM collagen and fibronectin influence diastolic stiffness. ECM binding to membrane-bound receptors, or integrins, directly links ECM to cardiac muscle and fibroblast cells, affording it the permissive role to modulate heart function. To better understand the ECM structure–function relationship in the old heart, we studied the relative protein content of these ECM proteins and integrins across three age groups. Old Balb-c mice (20 months) exhibit biventricular, cardiac hypertrophy, and greater left ventricular (LV) collagen, fibronectin, α1 and α5 integrin protein than middle-aged (12 months) or young (2 months) LV (P<0.05). β1 integrin protein content is lower in old LV (P<0.05). These data show that advancing age is associated with greater collagen, fibronectin, α1 and α5 integrin content, suggesting that these matrix proteins undergo coordinated regulation in the aging heart. The differential integrin and ECM protein content suggests that there is regulatory signaling to the fibroblasts, which maintain the cardiac ECM.

Introduction

Aging results in the progressive decline of the cardiovascular system, characterized in part by an increase in wall thickness of the ventricles (Spirduso, 1995; Molina et al., 1999). Aged rodents experience ventricular hypertrophy associated with an excess accumulation of the fibrillar protein collagen, a major structural component of the cardiac extracellular matrix (ECM) (Thomas et al., 1992; Lin and Bissell, 1993; Seccia et al., 1999). Within the cardiac ECM, the collagen network supports and aligns myocytes, blood vessels and lymphatic vessels relative to one another, and helps maintain myocardial mass and structure. Specifically, ECM collagen is the basis for myocardial diastolic stiffness and protects myocytes from overstretch (Beyar et al., 1993; Weber et al., 1994; MacKenna et al., 1996). When this relatively non-elastic material is expressed in the myocardium in disproportionate amounts, fibrosis occurs, along with a reduction in diastolic distensibility (Weber et al., 1994; Burgess et al., 1996). The occurrence of fibrosis increases with age (Lakatta, 1993a, Lakatta, 1993b), and creates a denser ECM with thicker perimysial tendons and strands of collagen (Eghbali et al., 1989). In cardiac fibrosis, collagen fibers radiate outward between muscle fibers, occupying and encompassing the interstitial space (Eghbali et al., 1989), and are a major cause of myocardial stiffness resulting in a decreased pumping and relaxation capacity of the heart (Brilla et al., 1990; Burgess et al., 1996; Villari et al., 1997). Fibrosis is found in non-diseased old hearts, and therefore can be independently associated with the aging process (Lakatta, 1993a, Lakatta, 1993b).

The laying-down of collagen in the cardiac ECM as a result of aging is prefaced by increased synthesis of the glycoprotein fibronectin. In development, and in response to stressors such as hypertension, fibronectin is the initial matrix upon which collagen is laid (Barry and Mosher, 1989). Fibronectin protein content is important in cell growth, development, integration, adhesion, cytoskeletal organization, tissue remodeling, cardiac morphogenesis, and possibly aging as well (Sabri et al., 1995). In both hypertrophic and dilated cardiomyopathy, mRNA expression for fibronectin increases with age (Masutomo et al., 1999). Fibronectin binds to components of the ECM other than collagen, such as membrane-bound integrins (Ruoslahti, 1991; Sheppard et al., 1994), which permit mechanical and chemical signaling to and from the ECM. The interaction between fibronectin, the ECM and other cells in the heart can alter the phenotype of the adhering cells by mediating changes in gene expression (Werb et al., 1989; Hedin et al., 1990). Additionally, the complex of structural components, including cytoskeletal, linkage, and extracellular proteins such as fibronectin, are involved in age-associated functional deterioration. Specifically, cytoskeletal abnormalities are significantly associated with indices of intrinsic contractile and diastolic dysfunction in aged, failing hearts (Heling et al., 2000). This strong structure–function relationship is observed in aged, non-diseased hearts as well (Ahumada and Saffitz, 1984).

Binding of fibronectin and other ECM proteins is regulated by integrins, which may be associated with age-related changes in protein content (Albelda and Buck, 1990). Integrins, a family of membrane receptors which play a fundamental role in the mediation of cell interactions, consist of a heterodimer of α and β chains linking cell:cell, cell:ECM and ECM:cytoskeleton contacts (Carver and Terracio, 1993; Rojas and Ahmed, 1999; Etzioni, 2000). Studies in the neonatal heart have found that the α1 and β1 integrins serve to transduce the phenotypic and positional information contained within the tertiary structure of an aligned collagen gel in vitro (Carver et al., 1994). It is increasingly apparent that integrins also transduce messages to the interior of the cell via classic signaling pathways, and impact upon such fundamental cellular processes, such as proliferation, apoptosis, differentiation, and motility. Indeed, dysregulation of these processes are a feature of many malignancies (Jones and Walker, 1999). In the adult heart, integrins are selectively expressed in response to their immediate cellular environment (Ashizawa et al., 1996; Booz & Baker, 1995; Burgess et al., unpublished data). The adaptability of cells to regulate their integrin protein content is thought to be crucial for cell function in development, disease and possibly aging.

Given the importance of collagen and fibronectin in the remodeling of the aged cardiac ECM, and their reliance on the integrins to bind and transmit information, this study tests the hypothesis that advancing age is associated with significantly altered protein content. Specifically, we hypothesize that the content of collagen and fibronectin is altered in advancing age, with a concomitant directional change in the α1, α2, α5 and β1 integrin protein in the left ventricle of the heart. The design of this work is primarily descriptive in that it carefully characterizes the age-associated changes that occur in the cardiac ECM, such that these changes may then be used as a springboard for future mechanistic research to elucidate the potential functional role(s) of age-associated matrix remodeling.