Contractility of myofibrils from the heart and diaphragm muscles measured with atomic force cantilevers: Effects of heart-specific deletion of arginyl-tRNA–protein transferase☆
Introduction
Protein arginylation is a post-translational cellular process catalyzed by arginyl-tRNA–protein transferase, responsible for transferring arginine from tRNA into proteins [8], [10], [17]. Arginylation is important for many aspects of cell and cardiovascular development [7]. We have recently developed a mouse knockout (KO) model with deletion of cardiac-specific arginyl-tRNA–protein transferase in differentiated cardiac myocytes, with α-myosin heavy chain promoter that induces dilated cardiomyopathy and leads to late postnatal lethality, with characteristics that are reminiscent of chronic heart failure (CHF) in humans [6]. These mice (α-MHCAte1) represent an appropriate model to investigate cardiomyopathy and CHF [7].
Analysis of cardiac myocytes isolated from complete and conditional KO of arginyl-tRNA–protein transferase mice reveals impairment in heart integrity, myofibril development and organization, as well as a disintegration of intercalated disks among myofibrils, symptoms that worsen with age [6], [17]. Furthermore, the α-MHCAte1 mice present impaired heart contractility, including a weakened contraction, arrhythmias, and fibrillations [6], [17]. A decreased contractility could result from fewer myosin cross-bridges attached to actin during activation, but it could also result from changes in the rate constants of cross-bridges when shifting from weakly- to strongly-bound states during the actomyosin cycle. The detailed contractile characteristics of α-MHCAte1 cardiac muscles have not been thoroughly investigated to date.
CHF is frequently associated with changes in skeletal muscle functioning, including peripheral and inspiratory muscle weakness [3], [4], [11], [12], [19], [20], [21], [23], [25] and an impaired passive elasticity of muscle fibers [3], [23]. Human CHF is frequently associated with skeletal muscle involvement, including peripheral and inspiratory muscles [19], [25]. Inspiratory muscle weakness may be present in more than 30% of patients in specialized CHF clinics, and has important impact in functional capacity, quality of life, and prognosis [5], [19], [25]. The mechanism responsible for the inspiratory muscle weakness has not been elucidated, but it has been associated with changes in fiber type and oxidative capacity [4], [20], intracellular calcium (Ca2 +) regulation [12], systemic neurohumoral activation and oxidative stress [11]. Taken together, these data suggest that an intrinsic contractile dysfunction of the diaphragm contributes to inspiratory muscle weakness in CHF.
One of the major difficulties in discriminating the mechanisms of muscle weakness in CHF is isolating the different components of muscle contraction in the whole heart or diaphragm, or in muscle bundle preparations. Sub-cellular mechanical analysis of cardiac and diaphragm muscles is not trivial, given the technical complexity of such experiments. Consequently, we lack information on the molecular sites of muscle weakness in CHF. In the present study, we used a newly developed system to measure contractile characteristics of isolated myofibrils from the heart and diaphragm using atomic force cantilevers (AFC) [9]. The system was equipped for biophysical analysis of striated muscles with high force (nN) and time resolution (μs), allowing analysis of molecular and subcellular contractile components of the myocardium and diaphragm in CHF. Notably, the use of myofibrils permits the investigation of the smallest contractile unit of these muscles—the sarcomeres—and effects from other cellular and extracellular sources (e.g. excitation–contraction coupling, action potential, membranes) are avoided. We used myofibrils from α-MHCAte1 to evaluate their length dependence of active and passive force production (normalized per cross-sectional area), and the rates of force development Kact, redevelopment (Ktr) and relaxation (Krel), parameters that provide information on myosin cross-bridge kinetics. We tested the specific hypothesis that myocardium and diaphragm contractile properties are impaired in α-MHCAte1.
Section snippets
Animals
α-MHCAte1 mice (n = 6) and age-matched wild type mice (WT) (n = 6) were used in this study. The mouse model with cardiomyocyte specific α-MHCAte1 was developed by using a previously developed “Ate1-floxed” mouse line, which was crossed with αMHC-Cre mice [1], in which Cre recombinase is expressed under cardiomyocyte-specific α-myosin heavy chain promoter that activates upon differentiation, resulting in Ate1 deletion in the heart muscle (see [6] for details of the mouse strain generation). The
Electron microscopy of heart and diaphragm muscles
Myofibrils from the hearts isolated from α-MHCAte1 mice showed visible defects, including disorganized sarcomeres, diffuse Z-bands, and poorly pronounced M-lines (Fig. 2). These abnormal patterns indicate defects in the structure of the myofibril components, resulting in displacement of some of the thick filaments from the center of the sarcomere. These displacements are common in sarcomeres with defective titin, a large scaffolding protein that plays a key role in sarcomere organization and
Discussion
The main purpose of this study was to use a newly developed system of AFC to measure the contractile properties of myofibrils isolated from the myocardium and diaphragm from α-MHCAte1 mice, an animal model for human cardiomyopathy [6], [7]. Furthermore, we tested the hypothesis that CHF is indirectly responsible for altered mechanical properties of the diaphragm muscle. In accordance with our previous study [6], myofibrils from the myocardium of α-MHCAte1 mice presented reduced active and
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Grant support: This study received financial support from the Canadian Institutes for Health Research (CIHR grant to D.E.R.), Canada, Fonds de Recherche du Québec—Nature et Technologies (FQRNT), Canada, and the National Institute of Health (NIH R01 HL084419 grant to A.K.), U.S.A. P.A.B. Ribeiro received scholarships from the Brazilian Coordination for the Development of Superior Education Personnel (CAPES) and from the Brazilian Research Council (CNPq), Brasília, Brazil. J.P. Ribeiro was an established investigator of the CNPq, Brasília, Brazil at the time of this study. J.P. Ribeiro passed away in August, 2012. The authors of this paper are grateful for his important contributions during the development of this study. The authors would like to thank Dr. Junling Wang and Tiberius Brastaviceanu for technical assistance with some of the experiments performed in this study.
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This author takes responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation.