Getting to the heart of cardiac remodeling; how collagen subtypes may contribute to phenotype

Abstract

The objective of this study was to investigate the nature and biomechanical properties of collagen fibers within the human myocardium. Targeting cardiac interstitial abnormalities will likely become a major focus of future preventative strategies with regard to the management of cardiac dysfunction. Current knowledge regarding the component structures of myocardial collagen networks is limited, further delineation of which will require application of more innovative technologies. We applied a novel methodology involving combined confocal laser scanning and atomic force microscopy to investigate myocardial collagen within ex-vivo right atrial tissue from 10 patients undergoing elective coronary bypass surgery. Immuno-fluorescent co-staining revealed discrete collagen I and III fibers. During single fiber deformation, overall median values of stiffness recorded in collagen III were 37 ± 16% lower than in collagen I [p < 0.001]. On fiber retraction, collagen I exhibited greater degrees of elastic recoil [p < 0.001; relative percentage increase in elastic recoil 7 ± 3%] and less energy dissipation than collagen III [p < 0.001; relative percentage increase in work recovered 7 ± 2%]. In atrial biopsies taken from patients in permanent atrial fibrillation (n = 5) versus sinus rhythm (n = 5), stiffness of both collagen fiber subtypes was augmented (p < 0.008). Myocardial fibrillar collagen fibers organize in a discrete manner and possess distinct biomechanical differences; specifically, collagen I fibers exhibit relatively higher stiffness, contrasting with higher susceptibility to plastic deformation and less energy efficiency on deformation with collagen III fibers. Augmented stiffness of both collagen fiber subtypes in tissue samples from patients with atrial fibrillation compared to those in sinus rhythm are consistent with recent published findings of increased collagen cross-linking in this setting.

Introduction

Collagen is the predominant protein in myocardial connective tissue and possesses versatile biomechanical properties that well-serve the complex structural and functional needs of the heart [1]. Specifically, its high stiffness resists excessive myocardial filling [2]; its high tensile strength confers resistance to cardiac rupture [3], [4]; while its ability to store and release elastic energy in a spring-like fashion contributes to myocardial re-lengthening, diastolic suction and optimal cardiac function [5], [6], [7].

Pathological alterations in myocardial collagen infrastructure, on the other hand, have many deleterious effects including altered myocardial stiffness and energy requirements, increased risk of arrhythmia, tethering and mechanical uncoupling of myocytes and impaired oxygen diffusion [1], [2]. Yet, current knowledge of the main component structures of myocardial collagen (molecular subtypes I and III) is surprisingly limited.

Firstly, the extent and nature of the interaction of collagen subtypes remains to be elucidated [3]. It is often presumed that these building blocks of myocardial supra-molecular fibrillar structures are organized therein as co-polymers [4]. However, much of the supporting data, oftentimes derived from non-human non-cardiac tissue, has dubious applicability to human myocardium, while the specificity of staining techniques often used to distinguish these subtypes has also been questioned [4], [5], [6].

A further issue that arises is whether the mechanical properties of collagen and thereby the myocardium are influenced by differential expression of one subtype of collagen over another [3], [7]. Biomarker data have suggested that shifts towards excess collagen I synthesis may be important with regard to increased myocardial stiffness in hypertensive heart disease or aortic stenosis [8]. Conversely, others have suggested that a shift towards excess collagen III synthesis is associated with reduced cardiac stiffness and cardiac dilatation [7], [9], [10].

In addition to changes in collagen subtype ratios, collagen quality can also be affected by post-translational modifications of collagen molecules particularly increased cross-linking. This mechanism has recently been reported in atrial tissue from patients with atrial fibrillation (AFib) in association with up-regulated lysyl oxidase expression [11]. How this impacts on fiber stiffness and further affects tissue mechanics has not yet been fully elucidated.

Given the potential variations in the interstitial response to different pathological stimuli, a more complete understanding of the component parts of the interstitium and their impact on tissue mechanics is critical as we strive to develop specific and effective therapies. Further delineation of the biophysical properties of myocardial tissue collagens may require more innovative technologies.

Atomic force microscopy (AFM) is a well-validated technique that has recently been used to investigate the biomechanical properties of individual collagen fibers [12]. Using a novel methodology involving subtype-specific antibodies to identify collagen I and III in human myocardium and the application of combined confocal AFM, we sought to examine how collagen subtypes are organized in the heart and to further define their biomechanical properties. In addition, we sought to examine whether recently reported alterations in collagen quality in association with AFib were evident at the individual fiber level.