Original articleRemodeling of the sarcomeric cytoskeleton in cardiac ventricular myocytes during heart failure and after cardiac resynchronization therapy
Introduction
The cytoskeleton is a complex intracellular network of proteins essential for determining the shape and mechanical properties of cells [1]. It also helps to coordinate the function of subcellular proteins in all cell types. These functions range from anchoring cellular organelles such as the Golgi apparatus, nuclei and mitochondria to transmission of extracellular signals and coordinating contraction [1], [2]. Cytoskeletal proteins form the sarcomeres, which are the basic contractile units of striated muscle cells. Within each sarcomere is a complex arrangement of proteins [2], [3], [4]. Each sarcomere is bounded by the Z-disk [5]. The Z-disk is crucial for maintaining sarcomeric structure and function. The main component of the Z-disk is the protein α-actinin, even though it accounts for less than 20% of Z-disk weight [4]. Αlpha-actinin is an anti-parallel homodimer that anchors the actin filaments, which are essential for contraction in myocytes. It was initially believed that α-actinin solely provided an actin binding site [6]. However, subsequent studies demonstrated that α-actinin links to several transmembrane receptors, regulatory proteins, adherens junctions, focal adhesion sites and stress fibers [6]. Further studies revealed that α-actinin plays a pivotal role in the assembly of sarcomeres and the regular arrangement of myofilaments [7], [8]. The studies suggest that sarcomere assembly is initiated by small Z-bodies comprising complexes of α-actinin and associated proteins. Subsequently, the Z-bodies expand, fuse and align in Z-bands. Similar as other proteins in the Z-disk α-actinin exhibits a surprisingly dynamic exchange with the cytosolic pool [8]. Exchange rates were larger in Z-bodies than Z-disks indicating that molecular interactions increase the stability of Z-disks. Four different isoforms of α-actinin exist. The genes ACTN-1 and ACTN-4 encode α-actinin isoforms that are expressed in non-muscle cells, where these isoforms of α-actinin contribute to the actin cytoskeleton. ACTN-2 and ACTN-3 encode isoforms specific to the Z-disks of sarcomeres found in striated muscle fibers [4], [9], [10], [11], with α-actinin-2 being the only cardiac specific isoform [4].
Remodeling of sarcomeric proteins in cardiac disease has been implicated in reduced ventricular function [2]. In particular, it has been suggested that the transition from hypertrophy to heart failure (HF) occurs in two consecutive stages. The first stage is reversible and involves an accumulation of cytoskeletal proteins to counteract the increased strain imposed on the myocardium. The latter stage becomes irreversible and is characterized by a loss of contractile filaments and crucial sarcomeric proteins, including α-actinin, titin, and myomesin. Several cardiac diseases have been associated with remodeling of α-actinin and the Z-disk [9], [10], [11], [12]. Melo et al. demonstrated that Trypanosoma cruzi in mouse myocytes caused α-actinin distributions to lose their periodic structure and to localize to focal adhesion sites [11]. Hein et al. found depositions of α-actinin-1 in failing myocardium [10], while Oxford et al. showed an accumulation of electron dense material around Z-lines in canines with arrhythmogenic cardiomyopathy. Cardiac disorders have also been linked to mutations in the gene responsible for α-actinin-2, specifically dilated cardiomyopathy [13] and hypertrophic cardiomyopathy [9].
Here we investigated remodeling of α-actinin in two models of HF, synchronous (SHF) and dyssynchronous heart failure (DHF), as well as in a model of cardiac resynchronization therapy (CRT). Approximately 40% of patients suffering from HF develop delays of ventricular electrical activation that result in a dyssynchronous mechanical contraction of the ventricles. The electrical dyssynchrony results in a widened QRS interval, which has been shown to be an independent predictor of mortality and sudden cardiac death [14]. CRT is an established clinical therapy to treat DHF. CRT resynchronizes ventricular mechanical and electrical activity via biventricular pacing and has been proven effective at improving quality of life and reducing mortality in about 55% of patients [15]. A number of studies showed that CRT is associated with restoration of cardiac structure and function, for instance, the transverse tubular system and excitation–contraction coupling [16] as well as the electrophysiological and contractile properties of myocytes [17], [18]. However, little is known about the reorganization of sarcomeric structures and associated protein distributions in DHF and CRT.
Our hypothesis is that DHF associated remodeling of sarcomeric organization is reversed after CRT. We used α-actinin as a marker of sarcomeric organization. We applied high-resolution three-dimensional confocal microscopy to image α-actinin distributions in ventricular tissues and cells. Analyses of image stacks allowed us to provide quantitative data on the structural arrangement of α-actinin and its remodeling. Our studies revealed alterations of the spatial regularity of α-actinin. We applied Fourier analysis to characterize remodeling of the spatial arrangement of α-actinin. Also, our studies revealed longitudinal depositions of α-actinin in HF. Using methods of pattern detection we quantified their occurrences. For further insights into the composition of the longitudinal depositions we investigated colocalization of α-actinin with sarcomeric proteins and measured the density of α-actinin based on super-resolution microscopy. To shed light on α-actinin expression in our animal models we performed gene expression analyses and Western blotting.
Section snippets
Animal model, tissue and isolated cell preparation
All procedures involving the handling of animals were approved by the Animal Care and Use Committees of the Johns Hopkins University and the University of Utah. Protocols complied with the published Guide for the Use and Care of Laboratory Animals published by the National Institutes of Health.
The applied animal models have been described previously [16], [19], [20], [21]. In previous studies, successful implementation of the DHF and CRT models was confirmed by increased and normalized QRS
Distribution of α-actinin in normal and heart failure tissue
We imaged sections from control canine cardiac ventricular tissue. A pre-processed 3D confocal microscopic image stack is shown in Fig. 1A. Pre-processing consisted of deconvolution, background removal and attenuation correction. The corresponding unprocessed image stack is presented in Fig. S4A. In these images, α-actinin was primarily arranged in regular, parallel, transverse sheets. These sheets ran laterally and exhibited frequent gaps and extended throughout the myocytes. This pattern is
Discussion
Our study used three-dimensional microscopy and image analyses to reveal previously unknown remodeling of sarcomeres in cardiac ventricular myocytes from failing hearts. Visual inspection of images exposed remodeling associated with HF, in particular the irregular arrangement and occurrence of longitudinal depositions of α-actinin. We applied Fourier analysis to measure the decrease of spatial regularity of the α-actinin distribution. In DHF cells, we found decreased regularity of α-actinin
Disclosures
Dr. Kass has served as a consultant to or on the advisory board of Boston Scientific Consulting. The other authors report no conflicts.
Acknowledgments
Sources of funding: The study has been supported by NIH grants R01 HL094464 (FBS) and PO1 HL077180 (DAK, GFT), and awards from the Nora Eccles Treadwell Foundation (JHB, FBS).
We thank Chris Hunter, Dr. Hui Li and Dr. Natalia Torres for their help and discussions. We also thank Dr. Manasa Gudheti (Vutara, Inc, USA) for her advice on the super-resolution microscopy studies. The titin antibody was a kind gift of Dr. Elisabeth Ehler, King's College, London, UK.
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